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Sample records for meiotic recombination dna

  1. Modulation of meiotic homologous recombination by DNA helicases.

    PubMed

    Lorenz, Alexander

    2016-12-08

    DNA helicases are ATP-driven motor proteins which translocate along DNA capable of dismantling DNA-DNA interactions and/or removing proteins bound to DNA. These biochemical capabilities make DNA helicases main regulators of crucial DNA metabolic processes, including DNA replication, DNA repair, and genetic recombination. This budding topic will focus on reviewing the function of DNA helicases important for homologous recombination during meiosis, and discuss recent advances in how these modulators of meiotic recombination are themselves regulated. The emphasis is placed on work in the two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, which has vastly expanded our understanding of meiotic homologous recombination, a process whose correct execution is instrumental for healthy gamete formation, and thus functioning sexual reproduction. Copyright © 2016 John Wiley & Sons, Ltd.

  2. A DNA topoisomerase VI-like complex initiates meiotic recombination.

    PubMed

    Vrielynck, Nathalie; Chambon, Aurélie; Vezon, Daniel; Pereira, Lucie; Chelysheva, Liudmila; De Muyt, Arnaud; Mézard, Christine; Mayer, Claudine; Grelon, Mathilde

    2016-02-26

    The SPO11 protein catalyzes the formation of meiotic DNA double strand breaks (DSBs) and is homologous to the A subunit of an archaeal topoisomerase (topo VI). Topo VI are heterotetrameric enzymes comprising two A and two B subunits; however, no topo VIB involved in meiotic recombination had been identified. We characterized a structural homolog of the archaeal topo VIB subunit [meiotic topoisomerase VIB-like (MTOPVIB)], which is essential for meiotic DSB formation. It forms a complex with the two Arabidopsis thaliana SPO11 orthologs required for meiotic DSB formation (SPO11-1 and SPO11-2) and is absolutely required for the formation of the SPO11-1/SPO11-2 heterodimer. These findings suggest that the catalytic core complex responsible for meiotic DSB formation in eukaryotes adopts a topo VI-like structure.

  3. RPA homologs and ssDNA processing during meiotic recombination.

    PubMed

    Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

    2016-06-01

    Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

  4. Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

    PubMed Central

    Najor, Nicole A.; Weatherford, Layne; Brush, George S.

    2016-01-01

    In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs) are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control. PMID:27678521

  5. Genetic controls of meiotic recombination and somatic DNA metabolism in Drosophila melanogaster.

    PubMed Central

    Baker, B S; Boyd, J B; Carpenter, A T; Green, M M; Nguyen, T D; Ripoll, P; Smith, P D

    1976-01-01

    Recombination-defective meiotic mutants and mutagen-sensitive mutants of D. melanogaster have been examined for their effects on meiotic chromosome behavior, sensitivity to killing by mutagens, somatic chromosome integrity, and DNA repair processes. Several loci have been identified that specify functions that are necessary for both meiotic recombination and DNA repair processes, whereas mutants at combination and DNA repair processes, whereas mutants at other loci appear to be defective in only one pathway of DNA processing. PMID:825857

  6. Connecting by breaking and repairing: mechanisms of DNA strand exchange in meiotic recombination.

    PubMed

    Sansam, Christopher L; Pezza, Roberto J

    2015-07-01

    During prophase of meiosis I, homologous chromosomes interact and undergo recombination. Successful completion of these processes is required in order for the homologous chromosomes to mount the meiotic spindle as a pair. The organization of the chromosomes into pairs ensures orderly segregation to opposite poles of the dividing cell, such that each gamete receives one copy of each chromosome. Chiasmata, the cytological manifestation of crossover products of recombination, physically connect the homologs in pairs, providing a linkage that facilitates their segregation. Consequently, mutations that reduce the level of recombination are invariably associated with increased errors in meiotic chromosome segregation. In this review, we focus on recent biochemical and genetic advances in elucidating the mechanisms of meiotic DNA strand exchange catalyzed by the Dmc1 protein. We also discuss the mode by which two recombination mediators, Hop2 and Mnd1, facilitate rate-limiting steps of DNA strand exchange catalyzed by Dmc1.

  7. Connecting by breaking and repairing: mechanisms of DNA strand exchange in meiotic recombination

    PubMed Central

    Sansam, Christopher L; Pezza, Roberto J

    2015-01-01

    During prophase of meiosis I, homologous chromosomes interact and undergo recombination. Successful completion of these processes is required in order for the homologous chromosomes to mount the meiotic spindle as a pair. The organization of the chromosomes into pairs ensures orderly segregation to opposite poles of the dividing cell, such that each gamete receives one copy of each chromosome. Chiasmata, the cytological manifestation of crossover products of recombination, physically connect the homologs in pairs, providing a linkage that facilitates their segregation. Consequently, mutations that reduce the level of recombination are invariably associated with increased errors in meiotic chromosome segregation. In this review, we focus on recent biochemical and genetic advances in elucidating the mechanisms of meiotic DNA strand exchange catalyzed by the Dmc1 protein. We also discuss the mode by which two recombination mediators, Hop2 and Mnd1, facilitate rate-limiting steps of DNA strand exchange catalyzed by Dmc1. PMID:25953379

  8. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  9. DNA methylation restrains transposons from adopting a chromatin signature permissive for meiotic recombination

    PubMed Central

    Zamudio, Natasha; Barau, Joan; Teissandier, Aurélie; Walter, Marius; Borsos, Maté; Servant, Nicolas; Bourc'his, Déborah

    2015-01-01

    DNA methylation is essential for protecting the mammalian germline against transposons. When DNA methylation-based transposon control is defective, meiotic chromosome pairing is consistently impaired during spermatogenesis: How and why meiosis is vulnerable to transposon activity is unknown. Using two DNA methylation-deficient backgrounds, the Dnmt3L and Miwi2 mutant mice, we reveal that DNA methylation is largely dispensable for silencing transposons before meiosis onset. After this, it becomes crucial to back up to a developmentally programmed H3K9me2 loss. Massive retrotransposition does not occur following transposon derepression, but the meiotic chromatin landscape is profoundly affected. Indeed, H3K4me3 marks gained over transcriptionally active transposons correlate with formation of SPO11-dependent double-strand breaks and recruitment of the DMC1 repair enzyme in Dnmt3L−/− meiotic cells, whereas these features are normally exclusive to meiotic recombination hot spots. Here, we demonstrate that DNA methylation restrains transposons from adopting chromatin characteristics amenable to meiotic recombination, which we propose prevents the occurrence of erratic chromosomal events. PMID:26109049

  10. DNA methylation restrains transposons from adopting a chromatin signature permissive for meiotic recombination.

    PubMed

    Zamudio, Natasha; Barau, Joan; Teissandier, Aurélie; Walter, Marius; Borsos, Maté; Servant, Nicolas; Bourc'his, Déborah

    2015-06-15

    DNA methylation is essential for protecting the mammalian germline against transposons. When DNA methylation-based transposon control is defective, meiotic chromosome pairing is consistently impaired during spermatogenesis: How and why meiosis is vulnerable to transposon activity is unknown. Using two DNA methylation-deficient backgrounds, the Dnmt3L and Miwi2 mutant mice, we reveal that DNA methylation is largely dispensable for silencing transposons before meiosis onset. After this, it becomes crucial to back up to a developmentally programmed H3K9me2 loss. Massive retrotransposition does not occur following transposon derepression, but the meiotic chromatin landscape is profoundly affected. Indeed, H3K4me3 marks gained over transcriptionally active transposons correlate with formation of SPO11-dependent double-strand breaks and recruitment of the DMC1 repair enzyme in Dnmt3L(-/-) meiotic cells, whereas these features are normally exclusive to meiotic recombination hot spots. Here, we demonstrate that DNA methylation restrains transposons from adopting chromatin characteristics amenable to meiotic recombination, which we propose prevents the occurrence of erratic chromosomal events.

  11. Meiotic recombination breakpoints are associated with open chromatin and enriched with repetitive DNA elements in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Meiotic recombination provides the framework for the genetic variation in natural and artificial populations of eukaryotes through the creation of novel haplotypes. Thus, determining the molecular characteristics of meiotic recombination remains essential for future plant breeding efforts, which hea...

  12. DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis.

    PubMed

    Yelina, Nataliya E; Lambing, Christophe; Hardcastle, Thomas J; Zhao, Xiaohui; Santos, Bruno; Henderson, Ian R

    2015-10-15

    During meiosis, homologous chromosomes undergo crossover recombination, which is typically concentrated in narrow hot spots that are controlled by genetic and epigenetic information. Arabidopsis chromosomes are highly DNA methylated in the repetitive centromeres, which are also crossover-suppressed. Here we demonstrate that RNA-directed DNA methylation is sufficient to locally silence Arabidopsis euchromatic crossover hot spots and is associated with increased nucleosome density and H3K9me2. However, loss of CG DNA methylation maintenance in met1 triggers epigenetic crossover remodeling at the chromosome scale, with pericentromeric decreases and euchromatic increases in recombination. We used recombination mutants that alter interfering and noninterfering crossover repair pathways (fancm and zip4) to demonstrate that remodeling primarily involves redistribution of interfering crossovers. Using whole-genome bisulfite sequencing, we show that crossover remodeling is driven by loss of CG methylation within the centromeric regions. Using cytogenetics, we profiled meiotic DNA double-strand break (DSB) foci in met1 and found them unchanged relative to wild type. We propose that met1 chromosome structure is altered, causing centromere-proximal DSBs to be inhibited from maturation into interfering crossovers. These data demonstrate that DNA methylation is sufficient to silence crossover hot spots and plays a key role in establishing domains of meiotic recombination along chromosomes.

  13. DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature

    PubMed Central

    Hyppa, Randy W.; Fowler, Kyle R.; Cipak, Lubos; Gregan, Juraj; Smith, Gerald R.

    2014-01-01

    Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant, pat1-as2, of the fission yeast Schizosaccharomyces pombe that undergoes synchronous meiosis at 25°C when an ATP analog is added to the culture. Here, we compare recombination intermediates in pat1-as2 at 25°C with those in the widely used pat1-114 temperature-sensitive mutant at 34°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25°C. In both conditions, Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination. PMID:24089141

  14. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    PubMed

    Becherel, Olivier J; Yeo, Abrey J; Stellati, Alissa; Heng, Evelyn Y H; Luff, John; Suraweera, Amila M; Woods, Rick; Fleming, Jean; Carrie, Dianne; McKinney, Kristine; Xu, Xiaoling; Deng, Chuxia; Lavin, Martin F

    2013-04-01

    Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  15. Initiation of meiotic recombination in chromatin structure.

    PubMed

    Yamada, Takatomi; Ohta, Kunihiro

    2013-08-01

    Meiotic homologous recombination is markedly activated during meiotic prophase to play central roles in faithful chromosome segregation and conferring genetic diversity to gametes. It is initiated by programmed DNA double-strand breaks (DSBs) by the conserved protein Spo11, and preferentially occurs at discrete sites called hotspots. Since the functions of Spo11 are influenced by both of local chromatin at hotspots and higher-order chromosome structures, formation of meiotic DSBs is under regulation of chromatin structure. Therefore, investigating features and roles of meiotic chromatin is crucial to elucidate the in vivo mechanism of meiotic recombination initiation. Recent progress in genome-wide chromatin analyses tremendously improved our understanding on this point, but many critical questions are left unaddressed. In this review, we summarize current knowledge in the field, and also discuss the future problems that must be solved to understand the role of chromatin structure in meiotic recombination.

  16. Rad3-Cds1 mediates coupling of initiation of meiotic recombination with DNA replication. Mei4-dependent transcription as a potential target of meiotic checkpoint.

    PubMed

    Ogino, Keiko; Masai, Hisao

    2006-01-20

    Premeiotic S-phase and meiotic recombination are known to be strictly coupled in Saccharomyces cerevisiae. However, the checkpoint pathway regulating this coupling has been largely unknown. In fission yeast, Rad3 is known to play an essential role in coordination of DNA replication and cell division during both mitotic growth and meiosis. Here we have examined whether the Rad3 pathway also regulates the coupling of DNA synthesis and recombination. Inhibition of premeiotic S-phase with hydroxyurea completely abrogates the progression of meiosis, including the formation of DNA double-strand breaks (DSBs). DSB formation is restored in rad3 mutant even in the presence of hydroxyurea, although repair of DSBs does not take place or is significantly delayed, indicating that the subsequent recombination steps may be still inhibited. Examination of the roles of downstream checkpoint kinases reveals that Cds1, but not Chk1 or Mek1, is required for suppression of DSB in the presence of hydroxyurea. Transcriptional induction of some rec+ genes essential for DSB occurs at a normal timing and to a normal level in the absence of DNA synthesis in both the wild-type and cds1delta cells. On the other hand, the transcriptional induction of the mei4+ transcription factor and cdc25+ phosphatase, which is significantly suppressed by hydroxyurea in the wild-type cells, occurs almost to a normal level in cds1delta cells even in the presence of hydroxyurea. These results show that the Rad3-Cds1 checkpoint pathway coordinates initiation of meiotic recombination and meiotic cell divisions with premeiotic DNA synthesis. Because mei4+ is known to be required for DSB formation and cdc25+ is required for activation of meiotic cell divisions, we propose an intriguing possibility that the Rad3-Cds1 meiotic checkpoint pathway may target transcription of these factors.

  17. Mechanism and Regulation of Meiotic Recombination Initiation

    PubMed Central

    Lam, Isabel; Keeney, Scott

    2015-01-01

    Meiotic recombination involves the formation and repair of programmed DNA double-strand breaks (DSBs) catalyzed by the conserved Spo11 protein. This review summarizes recent studies pertaining to the formation of meiotic DSBs, including the mechanism of DNA cleavage by Spo11, proteins required for break formation, and mechanisms that control the location, timing, and number of DSBs. Where appropriate, findings in different organisms are discussed to highlight evolutionary conservation or divergence. PMID:25324213

  18. Evolutionarily diverse determinants of meiotic DNA break and recombination landscapes across the genome

    PubMed Central

    Fowler, Kyle R.; Sasaki, Mariko; Milman, Neta

    2014-01-01

    Fission yeast Rec12 (Spo11 homolog) initiates meiotic recombination by forming developmentally programmed DNA double-strand breaks (DSBs). DSB distributions influence patterns of heredity and genome evolution, but the basis of the highly nonrandom choice of Rec12 cleavage sites is poorly understood, largely because available maps are of relatively low resolution and sensitivity. Here, we determined DSBs genome-wide at near-nucleotide resolution by sequencing the oligonucleotides attached to Rec12 following DNA cleavage. The single oligonucleotide size class allowed us to deeply sample all break events. We find strong evidence across the genome for differential DSB repair accounting for crossover invariance (constant cM/kb in spite of DSB hotspots). Surprisingly, about half of all crossovers occur in regions where DSBs occur at low frequency and are widely dispersed in location from cell to cell. These previously undetected, low-level DSBs thus play an outsized and crucial role in meiosis. We further find that the influence of underlying nucleotide sequence and chromosomal architecture differs in multiple ways from that in budding yeast. DSBs are not strongly restricted to nucleosome-depleted regions, as they are in budding yeast, but are nevertheless spatially influenced by chromatin structure. Our analyses demonstrate that evolutionarily fluid factors contribute to crossover initiation and regulation. PMID:25024163

  19. The role of DNA helicases and their interaction partners in genome stability and meiotic recombination in plants.

    PubMed

    Knoll, Alexander; Puchta, Holger

    2011-03-01

    DNA helicases are enzymes that are able to unwind DNA by the use of the energy-equivalent ATP. They play essential roles in DNA replication, DNA repair, and DNA recombination in all organisms. As homologous recombination occurs in somatic and meiotic cells, the same proteins may participate in both processes, albeit not necessarily with identical functions. DNA helicases involved in genome stability and meiotic recombination are the focus of this review. The role of these enzymes and their characterized interaction partners in plants will be summarized. Although most factors are conserved in eukaryotes, plant-specific features are becoming apparent. In the RecQ helicase family, Arabidopsis thaliana RECQ4A has been shown before to be the functional homologue of the well-researched baker's yeast Sgs1 and human BLM proteins. It was surprising to find that its interaction partners AtRMI1 and AtTOP3α are absolutely essential for meiotic recombination in plants, where they are central factors of a formerly underappreciated dissolution step of recombination intermediates. In the expanding group of anti-recombinases, future analysis of plant helicases is especially promising. While no FBH1 homologue is present, the Arabidopsis genome contains homologues of both SRS2 and RTEL1. Yeast and mammals, on the other hand. only possess homologues of either one or the other of these helicases. Plants also contain several other classes of helicases that are known from other organisms to be involved in the preservation of genome stability: FANCM is conserved with parts of the human Fanconi anaemia proteins, as are homologues of the Swi2/Snf2 family and of PIF1.

  20. Epigenetic control of meiotic recombination in plants.

    PubMed

    Yelina, Natasha; Diaz, Patrick; Lambing, Christophe; Henderson, Ian R

    2015-03-01

    Meiotic recombination is a deeply conserved process within eukaryotes that has a profound effect on patterns of natural genetic variation. During meiosis homologous chromosomes pair and undergo DNA double strand breaks generated by the Spo11 endonuclease. These breaks can be repaired as crossovers that result in reciprocal exchange between chromosomes. The frequency of recombination along chromosomes is highly variable, for example, crossovers are rarely observed in heterochromatin and the centromeric regions. Recent work in plants has shown that crossover hotspots occur in gene promoters and are associated with specific chromatin modifications, including H2A.Z. Meiotic chromosomes are also organized in loop-base arrays connected to an underlying chromosome axis, which likely interacts with chromatin to organize patterns of recombination. Therefore, epigenetic information exerts a major influence on patterns of meiotic recombination along chromosomes, genetic variation within populations and evolution of plant genomes.

  1. Meiotic recombination involving heterozygous large insertions in Saccharomyces cerevisiae: formation and repair of large, unpaired DNA loops.

    PubMed Central

    Kearney, H M; Kirkpatrick, D T; Gerton, J L; Petes, T D

    2001-01-01

    Meiotic recombination in Saccharomyces cerevisiae involves the formation of heteroduplexes, duplexes containing DNA strands derived from two different homologues. If the two strands of DNA differ by an insertion or deletion, the heteroduplex will contain an unpaired DNA loop. We found that unpaired loops as large as 5.6 kb can be accommodated within a heteroduplex. Repair of these loops involved the nucleotide excision repair (NER) enzymes Rad1p and Rad10p and the mismatch repair (MMR) proteins Msh2p and Msh3p, but not several other NER (Rad2p and Rad14p) and MMR (Msh4p, Msh6p, Mlh1p, Pms1p, Mlh2p, Mlh3p) proteins. Heteroduplexes were also formed with DNA strands derived from alleles containing two different large insertions, creating a large "bubble"; repair of this substrate was dependent on Rad1p. Although meiotic recombination events in yeast are initiated by double-strand DNA breaks (DSBs), we showed that DSBs occurring within heterozygous insertions do not stimulate interhomologue recombination. PMID:11514439

  2. Top3-Rmi1 DNA single-strand decatenase is integral to the formation and resolution of meiotic recombination intermediates.

    PubMed

    Kaur, Hardeep; De Muyt, Arnaud; Lichten, Michael

    2015-02-19

    The topoisomerase III (Top3)-Rmi1 heterodimer, which catalyzes DNA single-strand passage, forms a conserved complex with the Bloom's helicase (BLM, Sgs1 in budding yeast). This complex has been proposed to regulate recombination by disassembling double Holliday junctions in a process called dissolution. Top3-Rmi1 has been suggested to act at the end of this process, resolving hemicatenanes produced by earlier BLM/Sgs1 activity. We show here that, to the contrary, Top3-Rmi1 acts in all meiotic recombination functions previously associated with Sgs1, most notably as an early recombination intermediate chaperone, promoting regulated crossover and noncrossover recombination and preventing aberrant recombination intermediate accumulation. In addition, we show that Top3-Rmi1 has important Sgs1-independent functions that ensure complete recombination intermediate resolution and chromosome segregation. These findings indicate that Top3-Rmi1 activity is important throughout recombination to resolve strand crossings that would otherwise impede progression through both early steps of pathway choice and late steps of intermediate resolution.

  3. Solution structure and DNA-binding properties of the winged helix domain of the meiotic recombination HOP2 protein.

    PubMed

    Moktan, Hem; Guiraldelli, Michel F; Eyster, Craig A; Zhao, Weixing; Lee, Chih-Ying; Mather, Timothy; Camerini-Otero, R Daniel; Sung, Patrick; Zhou, Donghua H; Pezza, Roberto J

    2014-05-23

    The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination.

  4. Initiation of Meiotic Recombination in Mammals

    PubMed Central

    Kumar, Rajeev; de Massy, Bernard

    2010-01-01

    Meiotic recombination is initiated by the induction of programmed DNA double strand breaks (DSBs). DSB repair promotes homologous interactions and pairing and leads to the formation of crossovers (COs), which are required for the proper reductional segregation at the first meiotic division. In mammals, several hundred DSBs are generated at the beginning of meiotic prophase by the catalytic activity of SPO11. Currently it is not well understood how the frequency and timing of DSB formation and their localization are regulated. Several approaches in humans and mice have provided an extensive description of the localization of initiation events based on CO mapping, leading to the identification and characterization of preferred sites (hotspots) of initiation. This review presents the current knowledge about the proteins known to be involved in this process, the sites where initiation takes place, and the factors that control hotspot localization. PMID:24710101

  5. Meiotic recombination in mammals: localization and regulation.

    PubMed

    Baudat, Frédéric; Imai, Yukiko; de Massy, Bernard

    2013-11-01

    During meiosis, a programmed induction of DNA double-strand breaks (DSBs) leads to the exchange of genetic material between homologous chromosomes. These exchanges increase genome diversity and are essential for proper chromosome segregation at the first meiotic division. Recent findings have highlighted an unexpected molecular control of the distribution of meiotic DSBs in mammals by a rapidly evolving gene, PR domain-containing 9 (PRDM9), and genome-wide analyses have facilitated the characterization of meiotic DSB sites at unprecedented resolution. In addition, the identification of new players in DSB repair processes has allowed the delineation of recombination pathways that have two major outcomes, crossovers and non-crossovers, which have distinct mechanistic roles and consequences for genome evolution.

  6. Activation of an alternative, rec12 (spo11)-independent pathway of fission yeast meiotic recombination in the absence of a DNA flap endonuclease.

    PubMed

    Farah, Joseph A; Cromie, Gareth; Davis, Luther; Steiner, Walter W; Smith, Gerald R

    2005-12-01

    Spo11 or a homologous protein appears to be essential for meiotic DNA double-strand break (DSB) formation and recombination in all organisms tested. We report here the first example of an alternative, mutationally activated pathway for meiotic recombination in the absence of Rec12, the Spo11 homolog of Schizosaccharomyces pombe. Rad2, a FEN-1 flap endonuclease homolog, is involved in processing Okazaki fragments. In its absence, meiotic recombination and proper segregation of chromosomes were restored in rec12Delta mutants to nearly wild-type levels. Although readily detectable in wild-type strains, meiosis-specific DSBs were undetectable in recombination-proficient rad2Delta rec12Delta strains. On the basis of the biochemical properties of Rad2, we propose that meiotic recombination by this alternative (Rec*) pathway can be initiated by non-DSB lesions, such as nicks and gaps, which accumulate during premeiotic DNA replication in the absence of Okazaki fragment processing. We compare the Rec* pathway to alternative pathways of homologous recombination in other organisms.

  7. Meiotic Recombination: The Essence of Heredity.

    PubMed

    Hunter, Neil

    2015-10-28

    The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles. Thus, meiotic recombination underlies both the independent assortment of parental chromosomes and genetic linkage. This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotic recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase. A more in-depth review presents our understanding of how crossover and noncrossover pathways of meiotic recombination are differentiated and regulated. The final section of this review summarizes the studies that have defined defective recombination as a leading cause of pregnancy loss and congenital disease in humans.

  8. Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain.

    PubMed Central

    Kirkpatrick, D T; Fan, Q; Petes, T D

    1999-01-01

    The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent). PMID:10224246

  9. Spatiotemporal regulation of meiotic recombination by Liaisonin

    PubMed Central

    Miyoshi, Tomoichiro; Ito, Masaru; Ohta, Kunihiro

    2013-01-01

    Sexual reproduction involves diversification of genetic information in successive generations. Meiotic recombination, which substantially contributes to the increase in genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) catalyzed by the evolutionarily conserved Spo11 protein. Spo11 requires additional partner proteins for its DNA cleavage reaction. DSBs are preferentially introduced at defined chromosomal sites called “recombination hotspots.” Recent studies have revealed that meiotically established higher-order chromosome structures, such as chromosome axes and loops, are also crucial in the control of DSB formation. Most of the DSB sites are located within chromatin loop regions, while many of the proteins involved in DSB formation reside on chromosomal axes. Hence, DSB proteins and DSB sites seem to be distantly located. To resolve this paradox, we conducted comprehensive proteomics and ChIP-chip analyses on Spo11 partners in Schizosaccharomyces pombe, in combination with mutant studies. We identified two distinct DSB complexes, the “DSBC (DSB Catalytic core)“ and “SFT (Seven-Fifteen-Twenty four; Rec7-Rec15-Rec24)” subcomplexes. The DSBC subcomplex contains Spo11 and functions as the catalytic core for the DNA cleavage reaction. The SFT subcomplex is assumed to execute regulatory functions. To activate the DSBC subcomplex, the SFT subcomplex tethers hotspots to axes via its interaction with Mde2, which can interact with proteins in both DSBC and SFT subcomplexes. Thus, Mde2 is likely to bridge these two subcomplexes, forming a “tethered loop-axis complex.” It should be noted that Mde2 expression is strictly regulated by S phase checkpoint monitoring of the completion of DNA replication. From these observations, we proposed that Mde2 is a central coupler for meiotic recombination initiation to establish a tethered loop-axis complex in liaison with the S phase checkpoint. PMID:23572041

  10. Spatiotemporal regulation of meiotic recombination by Liaisonin.

    PubMed

    Miyoshi, Tomoichiro; Ito, Masaru; Ohta, Kunihiro

    2013-01-01

    Sexual reproduction involves diversification of genetic information in successive generations. Meiotic recombination, which substantially contributes to the increase in genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) catalyzed by the evolutionarily conserved Spo11 protein. Spo11 requires additional partner proteins for its DNA cleavage reaction. DSBs are preferentially introduced at defined chromosomal sites called "recombination hotspots." Recent studies have revealed that meiotically established higher-order chromosome structures, such as chromosome axes and loops, are also crucial in the control of DSB formation. Most of the DSB sites are located within chromatin loop regions, while many of the proteins involved in DSB formation reside on chromosomal axes. Hence, DSB proteins and DSB sites seem to be distantly located. To resolve this paradox, we conducted comprehensive proteomics and ChIP-chip analyses on Spo11 partners in Schizosaccharomyces pombe, in combination with mutant studies. We identified two distinct DSB complexes, the "DSBC (DSB Catalytic core)" and "SFT (Seven-Fifteen-Twenty four; Rec7-Rec15-Rec24)" subcomplexes. The DSBC subcomplex contains Spo11 and functions as the catalytic core for the DNA cleavage reaction. The SFT subcomplex is assumed to execute regulatory functions. To activate the DSBC subcomplex, the SFT subcomplex tethers hotspots to axes via its interaction with Mde2, which can interact with proteins in both DSBC and SFT subcomplexes. Thus, Mde2 is likely to bridge these two subcomplexes, forming a "tethered loop-axis complex." It should be noted that Mde2 expression is strictly regulated by S phase checkpoint monitoring of the completion of DNA replication. From these observations, we proposed that Mde2 is a central coupler for meiotic recombination initiation to establish a tethered loop-axis complex in liaison with the S phase checkpoint.

  11. An essential gene, ESR1, is required for mitotic cell growth, DNA repair and meiotic recombination in Saccharomyces cerevisiae.

    PubMed Central

    Kato, R; Ogawa, H

    1994-01-01

    A new mutant, which was sensitive to both methyl-methanesulfonate (MMS) and ultra-violet light (UV) and defective in meiotic recombination, was isolated from Saccharomyces cerevisiae. The gene, ESR1, was cloned by complementation of the MMS sensitivity of the mutant and found to be essential for cell growth, as the deleted haploid strain was lethal. The ESR1 gene was adjacent to the CKS1 gene on chromosome II and encoded a putative 2368-amino acid protein with a molecular weight of 273 k. The ESR1 transcript was 8.0 kb long and was induced during meiosis. The predicted Esr1 protein had a mosaic structure composed of homologous regions and showed amino acid sequence similarities to Schizosaccharomyces pombe rad3+ protein, which monitors completion of DNA repair synthesis, and cut1+ protein, which is required for spindle pole body (SPB) duplication. The Esr1 protein was also similar to phosphatidylinositol (PI) 3-kinases, including Saccharomyces cerevisiae TOR2 (and DRR1), which are involved in G1 progression. These results suggest that ESR1 is multi-functional throughout mitosis and meiosis. Images PMID:8065923

  12. An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination.

    PubMed Central

    Mackey, Z B; Ramos, W; Levin, D S; Walter, C A; McCarrey, J R; Tomkinson, A E

    1997-01-01

    Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase. PMID:9001252

  13. A DNA binding motif of meiotic recombinase Rec12 (Spo11) defined by essential glycine-202, and persistence of Rec12 protein after completion of recombination.

    PubMed

    DeWall, K Mark; Davidson, Mari K; Sharif, Wallace D; Wiley, Charla A; Wahls, Wayne P

    2005-08-15

    The Rec12 (Spo11) protein of the fission yeast Schizosaccharomyces pombe is a meiosis-specific ortholog of the catalytic subunit of type VI topoisomerases and is thought to catalyze double-strand DNA breaks that initiate recombination. We tested the hypothesis that the rec12-117 allele affects the choice of pathways by which recombination is resolved. DNA sequence analysis revealed a single missense mutation in the coding region (rec12-G202E). The corresponding glycine-202 residue of Rec12 protein is strictly conserved in proteins of the Rec12/Spo11/Top6A family. It maps to the base of the DNA binding pocket in the crystal structure of the archaeal ortholog, Top6A. The rec12-G202E mutants lacked crossover and non-crossover recombination, demonstrating that rec12-G202E does not affect choice of resolution pathway. Like rec12-D15 null mutants, the rec12-G202E mutants suffered chromosome segregation errors in meiosis I. The Rec12-G202E protein was as stable as wild-type Rec12, demonstrating that glycine-202 is essential for a biochemical activity of Rec12 protein, rather than for its stability. These findings suggest that Rec12 facilitates binding of the meiotic recombinase to its substrate, DNA. Interestingly, the bulk of Rec12 protein persisted until the time of anaphase I, and a portion of Rec12 protein persisted until the time of anaphase II, after which it was undetectable. This suggests that Rec12 protein has additional meiotic functions after completion of recombination in prophase, as inferred previously from genetic studies [Sharif, W.D., Glick, G.G., Davidson, M.K., Wahls, W.P., 2002. Distinct functions of S. pombe Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II. Cell Chromo. 1, 1].

  14. Meiotic and mitotic recombination in meiosis.

    PubMed

    Kohl, Kathryn P; Sekelsky, Jeff

    2013-06-01

    Meiotic crossovers facilitate the segregation of homologous chromosomes and increase genetic diversity. The formation of meiotic crossovers was previously posited to occur via two pathways, with the relative use of each pathway varying between organisms; however, this paradigm could not explain all crossovers, and many of the key proteins involved were unidentified. Recent studies that identify some of these proteins reinforce and expand the model of two meiotic crossover pathways. The results provide novel insights into the evolutionary origins of the pathways, suggesting that one is similar to a mitotic DNA repair pathway and the other evolved to incorporate special features unique to meiosis.

  15. A DNA binding motif of meiotic recombinase Rec12 (Spo11) defined by essential glycine-202, and persistence of Rec12 protein after completion of recombination

    PubMed Central

    DeWall, K. Mark; Davidson, Mari K.; Sharif, Wallace D.; Wiley, Charla A.; Wahls, Wayne P.

    2011-01-01

    The Rec12 (Spo11) protein of the fission yeast Schizosaccharomyces pombe is a meiosis-specific ortholog of the catalytic subunit of type VI topoisomerases and is thought to catalyze double-strand DNA breaks that initiate recombination. We tested the hypothesis that the rec12-117 allele affects the choice of pathways by which recombination is resolved. DNA sequence analysis revealed a single missense mutation in the coding region (rec12-G202E). The corresponding glycine-202 residue of Rec12 protein is strictly conserved in proteins of the Rec12/Spo11/Top6A family. It maps to the base of the DNA binding pocket in the crystal structure of the archaeal ortholog, Top6A. The rec12-G202E mutants lacked crossover and non-crossover recombination, demonstrating that rec12-G202E does not affect choice of resolution pathway. Like rec12-D15 null mutants, the rec12-G202E mutants suffered chromosome segregation errors in meiosis I. The Rec12-G202E protein was as stable as wild-type Rec12, demonstrating that glycine-202 is essential for a biochemical activity of Rec12 protein, rather than for its stability. These findings suggest that Rec12 facilitates binding of the meiotic recombinase to its substrate, DNA. Interestingly, the bulk of Rec12 protein persisted until the time of anaphase I, and a portion of Rec12 protein persisted until the time of anaphase II, after which it was undetectable. This suggests that Rec12 protein has additional meiotic functions after completion of recombination in prophase, as inferred previously from genetic studies. PMID:16009511

  16. Polyploidization increases meiotic recombination frequency in Arabidopsis

    PubMed Central

    2011-01-01

    Background Polyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination. Results Using two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations. Conclusions The evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence. PMID:21510849

  17. Meiotic recombination proteins localize to linear elements in Schizosaccharomyces pombe.

    PubMed

    Lorenz, Alexander; Estreicher, Anna; Kohli, Jürg; Loidl, Josef

    2006-08-01

    In fission yeast, meiotic prophase nuclei develop structures known as linear elements (LinEs), instead of a canonical synaptonemal complex. LinEs contain Rec10 protein. While Rec10 is essential for meiotic recombination, the precise role of LinEs in this process is unknown. Using in situ immunostaining, we show that Rec7 (which is required for meiosis-specific DNA double-strand break (DSB) formation) aggregates in foci on LinEs. The strand exchange protein Rad51, which is known to mark the sites of DSBs, also localizes to LinEs, although to a lesser degree. The number of Rec7 foci corresponds well with the average number of genetic recombination events per meiosis suggesting that Rec7 marks the sites of recombination. Rec7 and Rad51 foci do not co-localize, presumably because they act sequentially on recombination sites. The localization of Rec7 is dependent on Rec10 but independent of the DSB-inducing protein Rec12/Spo11. Neither Rec7 nor Rad51 localization depends on the LinE-associated proteins Hop1 and Mek1, but the formation of Rad51 foci depends on Rec10, Rec7, and, as expected, Rec12/Spo11. We propose that LinEs form around designated recombination sites before the induction of DSBs and that most, if not all, meiotic recombination initiates within the setting provided by LinEs.

  18. Indistinguishable Landscapes of Meiotic DNA Breaks in rad50+ and rad50S Strains of Fission Yeast Revealed by a Novel rad50+ Recombination Intermediate

    PubMed Central

    Hyppa, Randy W.; Cromie, Gareth A.; Smith, Gerald R.

    2008-01-01

    The fission yeast Schizosaccharomyces pombe Rec12 protein, the homolog of Spo11 in other organisms, initiates meiotic recombination by creating DNA double-strand breaks (DSBs) and becoming covalently linked to the DNA ends of the break. This protein–DNA linkage has previously been detected only in mutants such as rad50S in which break repair is impeded and DSBs accumulate. In the budding yeast Saccharomyces cerevisiae, the DSB distribution in a rad50S mutant is markedly different from that in wild-type (RAD50) meiosis, and it was suggested that this might also be true for other organisms. Here, we show that we can detect Rec12-DNA linkages in Sc. pombe rad50+ cells, which are proficient for DSB repair. In contrast to the results from Sa. cerevisiae, genome-wide microarray analysis of Rec12-DNA reveals indistinguishable meiotic DSB distributions in rad50+ and rad50S strains of Sc. pombe. These results confirm our earlier findings describing the occurrence of widely spaced DSBs primarily in large intergenic regions of DNA and demonstrate the relevance and usefulness of fission yeast studies employing rad50S. We propose that the differential behavior of rad50S strains reflects a major difference in DSB regulation between the two species—specifically, the requirement for the Rad50-containing complex for DSB formation in budding yeast but not in fission yeast. Use of rad50S and related mutations may be a useful method for DSB analysis in other species. PMID:19023408

  19. Meiotic recombination cold spots in chromosomal cohesion sites.

    PubMed

    Ito, Masaru; Kugou, Kazuto; Fawcett, Jeffrey A; Mura, Sachiko; Ikeda, Sho; Innan, Hideki; Ohta, Kunihiro

    2014-05-01

    Meiotic chromosome architecture called 'axis-loop structures' and histone modifications have been shown to regulate the Spo11-dependent formation of DNA double-strand breaks (DSBs) that trigger meiotic recombination. Using genome-wide chromatin immunoprecipitation (ChIP) analyses followed by deep sequencing, we compared the genome-wide distribution of the axis protein Rec8 (the kleisin subunit of meiotic cohesin) with that of oligomeric DNA covalently bound to Spo11, indicative of DSB sites. The frequency of DSB sites is overall constant between Rec8 binding sites. However, DSB cold spots are observed in regions spanning ±0.8 kb around Rec8 binding sites. The axis-associated cold spots are not due to the exclusion of Spo11 localization from the axis, because ChIP experiments showed that substantial Spo11 persists at Rec8 binding sites during DSB formation. Spo11 fused with Gal4 DNA binding domain (Gal4BD-Spo11) tethered in close proximity (≤0.8 kb) to Rec8 binding sites hardly forms meiotic DSBs, in contrast with other regions. In addition, H3K4 trimethylation (H3K4me3) remarkably decreases at Rec8 binding sites. These results suggest that reduced histone H3K4me3 in combination with inactivation of Spo11 activity on the axis discourages DSB hot spot formation.

  20. Meiotic recombination initiated by a double-strand break in rad50{Delta} yeast cells otherwise unable to initiate meiotic recombination

    SciTech Connect

    Malkova, A.; Haber, J.E.; Dawson, D.

    1996-06-01

    Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast`s 16 chromosomes. In Rad{sup +} strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad{sup +} and in rad50{Delta} cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50{Delta} cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad50{Delta} diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination. 57 refs., 5 figs., 3 tabs.

  1. Scaling and fractal behaviour underlying meiotic recombination.

    PubMed

    Waxman, D; Stoletzki, N

    2010-01-01

    In this paper we investigate some of the mathematical properties of meiotic recombination. Working within the framework of a genetic model with n loci, where alpha alleles are possible at each locus, we find that the proportion of all possible diploid parental genotypes that can produce a particular haploid gamete is exp[-n log(alpha(2)/[2alpha-1])]. We show that this proportion connects recombination with a fractal geometry of dimension log(2alpha-1)/log(alpha). The fractal dimension of a geometric object manifests itself when it is measured at increasingly smaller length scales. Decreasing the length scale of a geometric object is found to be directly analogous, in a genetics problem, to specifying a multilocus haplotype at a larger number of loci, and it is here that the fractal dimension reveals itself.

  2. Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination.

    PubMed

    Grey, Corinne; Barthès, Pauline; Chauveau-Le Friec, Gaëlle; Langa, Francina; Baudat, Frédéric; de Massy, Bernard

    2011-10-01

    Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.

  3. DNA recombination. Recombination initiation maps of individual human genomes.

    PubMed

    Pratto, Florencia; Brick, Kevin; Khil, Pavel; Smagulova, Fatima; Petukhova, Galina V; Camerini-Otero, R Daniel

    2014-11-14

    DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here, we present high-resolution maps of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors may dictate the efficiency of DSB formation. We find evidence for both GC-biased gene conversion and mutagenesis around meiotic DSB hotspots, while frequent colocalization of DSB hotspots with chromosome rearrangement breakpoints implicates the aberrant repair of meiotic DSBs in genomic disorders. Furthermore, our data indicate that DSB frequency is a major determinant of crossover rate. These maps provide new insights into the regulation of meiotic recombination and the impact of meiotic recombination on genome function.

  4. Proteins involved in meiotic recombination: a role in male infertility?

    PubMed

    Sanderson, Matthew L; Hassold, Terry J; Carrell, Douglas T

    2008-01-01

    Meiotic recombination results in the formation of crossovers, by which genetic information is exchanged between homologous chromosomes during prophase I of meiosis. Recombination is a complex process involving many proteins. Alterations in the genes involved in recombination may result in infertility. Molecular studies have improved our understanding of the roles and mechanisms of the proteins and protein complexes involved in recombination, some of which have function in mitotic cells as well as meiotic cells. Human gene sequencing studies have been performed for some of these genes and have provided further information on the phenotypes observed in some infertile individuals. However, further studies are needed to help elucidate the particular role(s) of a given protein and to increase our understanding of these protein systems. This review will focus on our current understanding of proteins involved in meiotic recombination from a genomic perspective, summarizing our current understanding of known mutations and single nucleotide polymorphisms that may affect male fertility by altering meiotic recombination.

  5. Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse.

    PubMed

    Balcova, Maria; Faltusova, Barbora; Gergelits, Vaclav; Bhattacharyya, Tanmoy; Mihola, Ondrej; Trachtulec, Zdenek; Knopf, Corinna; Fotopulosova, Vladana; Chvatalova, Irena; Gregorova, Sona; Forejt, Jiri

    2016-04-01

    Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies.

  6. Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse

    PubMed Central

    Balcova, Maria; Faltusova, Barbora; Gergelits, Vaclav; Bhattacharyya, Tanmoy; Mihola, Ondrej; Trachtulec, Zdenek; Knopf, Corinna; Fotopulosova, Vladana; Chvatalova, Irena; Gregorova, Sona; Forejt, Jiri

    2016-01-01

    Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies. PMID:27104744

  7. The Meiotic Recombination Checkpoint Suppresses NHK-1 Kinase to Prevent Reorganisation of the Oocyte Nucleus in Drosophila

    PubMed Central

    Lancaster, Oscar M.; Breuer, Manuel; Cullen, C. Fiona; Ito, Takashi; Ohkura, Hiroyuki

    2010-01-01

    The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired. PMID:21060809

  8. The Saccharomyces cerevisiae RDN1 locus is sequestered from interchromosomal meiotic ectopic recombination in a SIR2-dependent manner.

    PubMed Central

    Davis, E S; Shafer, B K; Strathern, J N

    2000-01-01

    Meiotic ectopic recombination occurs at similar frequencies among many sites in the yeast genome, suggesting that all loci are similarly accessible to homology searching. In contrast, we found that his3 sequences integrated in the RDN1 (rDNA) locus were unusually poor participants in meiotic recombination with his3 sequences at other sites. We show that the low rate of meiotic ectopic recombination resulted from the poor ability of RDN1::his3 to act as a donor sequence. SIR2 partially repressed interchromosomal meiotic ectopic recombination at RDN1, consistent with its role in regulating recombination, gene expression, and retrotransposition within RDN1. We propose that RDN1 is physically sequestered from meiotic homology searching mechanisms. PMID:10880466

  9. Functional interactions between SPO11 and REC102 during initiation of meiotic recombination in Saccharomyces cerevisiae.

    PubMed

    Kee, Kehkooi; Keeney, Scott

    2002-01-01

    In Saccharomyces cerevisiae, formation of the DNA double-strand breaks (DSBs) that initiate meiotic recombination requires the products of at least 10 genes. Spo11p is thought to be the catalytic subunit of the DNA cleaving activity, but the roles of the other proteins, and the interactions among them, are not well understood. This study demonstrates genetic and physical interactions between the products of SPO11 and another early meiotic gene required for DSB formation, REC102. We found that epitope-tagged versions of SPO11 and REC102 that by themselves were capable of supporting normal or nearly normal levels of meiotic recombination conferred a severe synthetic cold-sensitive phenotype when combined in the same cells. DSB formation, meiotic gene conversion, and spore viability were drastically reduced in the doubly tagged strain at a nonpermissive temperature. This conditional defect could be partially rescued by expression of untagged SPO11, but not by expression of untagged REC102, indicating that tagged REC102 is fully dominant for this synthetic phenotype. Both tagged and wild-type Spo11p co-immunoprecipitated with tagged Rec102p from meiotic cell extracts, indicating that these proteins are present in a common complex in vivo. Tagged Rec102p localized to the nucleus in whole cells and to chromatin on spread meiotic chromosomes. Our results are consistent with the idea that a multiprotein complex that includes Spo11p and Rec102p promotes meiotic DSB formation.

  10. Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination

    PubMed Central

    Lorenz, Alexander; Mehats, Alizée; Osman, Fekret; Whitby, Matthew C.

    2014-01-01

    During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved. PMID:25414342

  11. Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination.

    PubMed

    Lorenz, Alexander; Mehats, Alizée; Osman, Fekret; Whitby, Matthew C

    2014-12-16

    During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved.

  12. The Mek1 phosphorylation cascade plays a role in meiotic recombination of Schizosaccharomyces pombe

    PubMed Central

    Ohtaka, Ayami; Okuzaki, Daisuke; Saito, Takamune T; Russell, Paul

    2010-01-01

    Mek1 is a Chk2/Rad53/Cds1-related protein kinase that is required for proper meiotic progression of Schizosaccharomyces pombe. However, the molecular mechanisms of Mek1 regulation and Mek1 phosphorylation targets are unclear. Here, we report that Mek1 is phosphorylated at serine-12 (S12), S14 and threonine-15 (T15) by Rad3 (ATR) and/or Tel1 (ATM) kinases that are activated by meiotic programmed double-strand breaks (DSBs). Mutations of these sites by alanine replacement caused abnormal meiotic progression and recombination rates. Phosphorylation of these sites triggers autophosphorylation of Mek1; indeed, alanine replacement mutations of Mek1-T318 and -T322 residues in the activation loop of Mek1 reduced Mek1 kinase activity and meiotic recombination rates. Substrates of Mek1 include Mus81-T275, Rdh54-T6 and Rdh54-T673. Mus81-T275 is known to regulate the Mus81 function in DNA cleavage, whereas Rdh54-T6A/T673A mutant cells showed abnormal meiotic recombination. Taken together, we conclude that the phosphorylation of Mek1 by Rad3 or Tel1, Mek1 autophosphorylation and Mus81 or Rdh54 phosphorylation by Mek1 regulate meiotic progression in S. pombe. PMID:21084840

  13. Competition between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces Cerevisiae

    PubMed Central

    Fan, Q. Q.; Xu, F.; White, M. A.; Petes, T. D.

    1997-01-01

    In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes. PMID:9055076

  14. Interplay between modifications of chromatin and meiotic recombination hotspots.

    PubMed

    Brachet, Elsa; Sommermeyer, Vérane; Borde, Valérie

    2012-02-01

    Meiotic recombination lies at the heart of sexual reproduction. It is essential for producing viable gametes with a normal haploid genomic content and its dysfunctions can be at the source of aneuploidies, such as the Down syndrome, or many genetic disorders. Meiotic recombination also generates genetic diversity that is transmitted to progeny by shuffling maternal and paternal alleles along chromosomes. Recombination takes place at non-random chromosomal sites called 'hotspots'. Recent evidence has shown that their location is influenced by properties of chromatin. In addition, many studies in somatic cells have highlighted the need for changes in chromatin dynamics to allow the process of recombination. In this review, we discuss how changes in the chromatin landscape may influence the recombination map, and reciprocally, how recombination events may lead to epigenetic modifications at sites of recombination, which could be transmitted to progeny.

  15. Allele-dependent recombination frequency: homology requirement in meiotic recombination at the hot spot in the mouse major histocompatibility complex.

    PubMed

    Yoshino, M; Sagai, T; Lindahl, K F; Toyoda, Y; Moriwaki, K; Shiroishi, T

    1995-05-20

    Meiotic recombination break joints in the mouse major histocompatibility complex (MHC) are clustered within short segments known as hot spots. We systematically investigated the requirement for sequence homology between two chromosomes for recombination activity at the hot spot next to the Lmp2 gene. The results indicated that a high rate of recombination required a high degree of similarity of overall genome structure at the hot spot. In particular, the same copy number of repetitive sequences within the hot spot was essential for a high frequency of recombination, suggesting that recombination in mouse meiosis is more sensitive to heterozygous deletion or insertion of DNA than to mismatches of single-base substitutions.

  16. Meiotic recombination in Arabidopsis is catalysed by DMC1, with RAD51 playing a supporting role.

    PubMed

    Da Ines, Olivier; Degroote, Fabienne; Goubely, Chantal; Amiard, Simon; Gallego, Maria E; White, Charles I

    2013-01-01

    Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis.

  17. Meiotic recombination, synapsis, meiotic inactivation and sperm aneuploidy in a chromosome 1 inversion carrier.

    PubMed

    Kirkpatrick, Gordon; Chow, Victor; Ma, Sai

    2012-01-01

    Disrupted meiotic behaviour of inversion carriers may be responsible for suboptimal sperm parameters in these carriers. This study investigated meiotic recombination, synapsis, transcriptional silencing and chromosome segregation effects in a pericentric inv(1) carrier. Recombination (MLH1), synapsis (SYCP1, SYCP3) and transcriptional inactivation (γH2AX, BRCA1) were examined by fluorescence immunostaining. Chromosome specific rates of recombination were determined by fluorescence in-situ hybridization. Furthermore, testicular sperm was examined for aneuploidy and segregation of the inv(1). Our findings showed that global recombination rates were similar to controls. Recombination on the inv(1) and the sex chromosomes were reduced. The inv(1) associated with the XY body in 43.4% of cells, in which XY recombination was disproportionately absent, and 94.3% of cells displayed asynapsed regions which displayed meiotic silencing regardless of their association with the XY body. Furthermore, a low frequency of chromosomal imbalance was observed in spermatozoa (3.4%). Our results suggest that certain inversion carriers may display unimpaired global recombination and impaired recombination on the involved and the sex chromosomes during meiosis. Asynapsis or inversion-loop formation in the inverted region may be responsible for impaired spermatogenesis and may prevent sperm-chromosome imbalance.

  18. Multiple modes of chromatin configuration at natural meiotic recombination hot spots in fission yeast.

    PubMed

    Hirota, Kouji; Steiner, Walter W; Shibata, Takehiko; Ohta, Kunihiro

    2007-11-01

    The ade6-M26 meiotic recombination hot spot of fission yeast is defined by a cyclic AMP-responsive element (CRE)-like heptanucleotide sequence, 5'-ATGACGT-3', which acts as a binding site for the Atf1/Pcr1 heterodimeric transcription factor required for hot spot activation. We previously demonstrated that the local chromatin around the M26 sequence motif alters to exhibit higher sensitivity to micrococcal nuclease before the initiation of meiotic recombination. In this study, we have examined whether or not such alterations in chromatin occur at natural meiotic DNA double-strand break (DSB) sites in Schizosaccharomyces pombe. At one of the most prominent DSB sites, mbs1 (meiotic break site 1), the chromatin structure has a constitutively accessible configuration at or near the DSB sites. The establishment of the open chromatin state and DSB formation are independent of the CRE-binding transcription factor, Atf1. Analysis of the chromatin configuration at CRE-dependent DSB sites revealed both differences from and similarities to mbs1. For example, the tdh1+ locus, which harbors a CRE consensus sequence near the DSB site, shows a meiotically induced open chromatin configuration, similar to ade6-M26. In contrast, the cds1+ locus is similar to mbs1 in that it exhibits a constitutive open configuration. Importantly, Atf1 is required for the open chromatin formation in both tdh1+ and cds1+. These results suggest that CRE-dependent meiotic chromatin changes are intrinsic processes related to DSB formation in fission yeast meiosis. In addition, the results suggest that the chromatin configuration in natural meiotic recombination hot spots can be classified into at least three distinct categories: (i) an Atf1-CRE-independent constitutively open chromatin configuration, (ii) an Atf1-CRE-dependent meiotically induced open chromatin configuration, and (iii) an Atf1-CRE-dependent constitutively open chromatin configuration.

  19. Impact of histone H4K16 acetylation on the meiotic recombination checkpoint in Saccharomyces cerevisiae

    PubMed Central

    Cavero, Santiago; Herruzo, Esther; Ontoso, David; San-Segundo, Pedro A.

    2016-01-01

    In meiotic cells, the pachytene checkpoint or meiotic recombination checkpoint is a surveillance mechanism that monitors critical processes, such as recombination and chromosome synapsis, which are essential for proper distribution of chromosomes to the meiotic progeny. Failures in these processes lead to the formation of aneuploid gametes. Meiotic recombination occurs in the context of chromatin; in fact, the histone methyltransferase Dot1 and the histone deacetylase Sir2 are known regulators of the pachytene checkpoint in Saccharomyces cerevisiae. We report here that Sas2-mediated acetylation of histone H4 at lysine 16 (H4K16ac), one of the Sir2 targets, modulates meiotic checkpoint activity in response to synaptonemal complex defects. We show that, like sir2, the H4-K16Q mutation, mimicking constitutive acetylation of H4K16, eliminates the delay in meiotic cell cycle progression imposed by the checkpoint in the synapsis-defective zip1 mutant. We also demonstrate that, like in dot1, zip1-induced phosphorylation of the Hop1 checkpoint adaptor at threonine 318 and the ensuing Mek1 activation are impaired in H4-K16 mutants. However, in contrast to sir2 and dot1, the H4-K16R and H4-K16Q mutations have only a minor effect in checkpoint activation and localization of the nucleolar Pch2 checkpoint factor in ndt80-prophase-arrested cells. We also provide evidence for a cross-talk between Dot1-dependent H3K79 methylation and H4K16ac and show that Sir2 excludes H4K16ac from the rDNA region on meiotic chromosomes. Our results reveal that proper levels of H4K16ac orchestrate this meiotic quality control mechanism and that Sir2 impinges on additional targets to fully activate the checkpoint. PMID:28357333

  20. Shu1 Promotes Homolog Bias of Meiotic Recombination in Saccharomyces cerevisiae

    PubMed Central

    Hong, Soogil; Kim, Keun Pil

    2013-01-01

    Homologous recombination occurs closely between homologous chromatids with highly ordered recombinosomes through RecA homologs and mediators. The present study demonstrates this relationship during the period of “partner choice” in yeast meiotic recombination. We have examined the formation of recombination intermediates in the absence or presence of Shu1, a member of the PCSS complex, which also includes Psy3, Csm2, and Shu2. DNA physical analysis indicates that Shu1 is essential for promoting the establishment of homolog bias during meiotic homologous recombination, and the partner choice is switched by Mek1 kinase activity. Furthermore, Shu1 promotes both crossover (CO) and non-crossover (NCO) pathways of meiotic recombination. The inactivation of Mek1 kinase allows for meiotic recombination to progress efficiently, but is lost in homolog bias where most double-strand breaks (DSBs) are repaired via stable intersister joint molecules. Moreover, the Srs2 helicase deletion cells in the budding yeast show slightly reduced COs and NCOs, and Shu1 promotes homolog bias independent of Srs2. Our findings reveal that Shu1 and Mek1 kinase activity have biochemically distinct roles in partner choice, which in turn enhances the understanding of the mechanism associated with the precondition for homolog bias. PMID:24213600

  1. PRDM9 is a major determinant of meiotic recombination hotspots in humans and mice.

    PubMed

    Baudat, F; Buard, J; Grey, C; Fledel-Alon, A; Ober, C; Przeworski, M; Coop, G; de Massy, B

    2010-02-12

    Meiotic recombination events cluster into narrow segments of the genome, defined as hotspots. Here, we demonstrate that a major player for hotspot specification is the Prdm9 gene. First, two mouse strains that differ in hotspot usage are polymorphic for the zinc finger DNA binding array of PRDM9. Second, the human consensus PRDM9 allele is predicted to recognize the 13-mer motif enriched at human hotspots; this DNA binding specificity is verified by in vitro studies. Third, allelic variants of PRDM9 zinc fingers are significantly associated with variability in genome-wide hotspot usage among humans. Our results provide a molecular basis for the distribution of meiotic recombination in mammals, in which the binding of PRDM9 to specific DNA sequences targets the initiation of recombination at specific locations in the genome.

  2. Meiotic Recombination in Schizosaccharomyces pombe: A Paradigm for Genetic and Molecular Analysis

    PubMed Central

    Cromie, Gareth; Smith, Gerald R.

    2009-01-01

    The fission yeast Schizosaccharomyces pombe is especially well-suited for both genetic and biochemical analysis of meiotic recombination. Recent studies have revealed ~50 gene products and two DNA intermediates central to recombination, which we place into a pathway from parental to recombinant DNA. We divide recombination into three stages – chromosome alignment accompanying nuclear “horsetail” movement, formation of DNA breaks, and repair of those breaks – and we discuss the roles of the identified gene products and DNA intermediates in these stages. Although some aspects of recombination are similar to those in the distantly related budding yeast Saccharomyces cerevisiae, other aspects are distinctly different. In particular, many proteins required for recombination in one species have no clear ortholog in the other, and the roles of identified orthologs in regulating recombination often differ. Furthermore, in S. pombe the dominant joint DNA molecule intermediates contain single Holliday junctions, and intersister joint molecules are more frequent than interhomolog types, whereas in S. cerevisiae interhomolog double Holliday junctions predominate. We speculate that meiotic recombination in other organisms shares features of each of these yeasts. PMID:20157622

  3. Brca2-Pds5 complexes mobilize persistent meiotic recombination sites to the nuclear envelope.

    PubMed

    Kusch, Thomas

    2015-02-15

    Homologous recombination is required for reciprocal exchange between homologous chromosome arms during meiosis. Only select meiotic recombination events become chromosomal crossovers; the majority of recombination outcomes are noncrossovers. Growing evidence suggests that crossovers are repaired after noncrossovers. Here, I report that persisting recombination sites are mobilized to the nuclear envelope of Drosophila pro-oocytes during mid-pachytene. Their number correlates with the average crossover rate per meiosis. Proteomic and interaction studies reveal that the recombination mediator Brca2 associates with lamin and the cohesion factor Pds5 to secure persistent recombination sites at the nuclear envelope. In Rad51(-/-) females, all persistent DNA breaks are directed to the nuclear envelope. By contrast, a reduction of Pds5 or Brca2 levels abolishes the movement and has a negative impact on crossover rates. The data suggest that persistent meiotic DNA double-strand breaks might correspond to crossovers, which are mobilized to the nuclear envelope for their repair. The identification of Brca2-Pds5 complexes as key mediators of this process provides a first mechanistic explanation for the contribution of lamins and cohesins to meiotic recombination.

  4. Meiotic recombination at the Lmp2 hotspot tolerates minor sequence divergence between homologous chromosomes

    SciTech Connect

    Yoshino, Masayasu; Sagai, Tomoko; Shiroishi, Toshihiko

    1996-06-01

    Recombination is widely considered to linearly depend on the length of the homologous sequences. An 11% mismatch decreases the rate of phage-plasmid recombination 240-fold. Two single nucleotide mismatches, which reduce the longest uninterrupted stretch of similarity from 232 base pairs (bp) to 134 bp, reduce gene conversion in mouse L cells 20-fold. The efficiency of gene targeting through homologous recombination in mouse embryonic stem cells can be increased by using an isogenic, rather than a non-isogenic, DNA construct. In this study we asked whether a high degree of sequence identity between homologous mouse chromosomes enhances meiotic recombination at a hotspot. Sites of meiotic recombination in the mouse major histocompatibility complex (MHC) class II region are not randomly distributed but are almost all clustered within short segments known as recombinational hotspots. The wm7 MHC haplotype, derived from Japanese wild mice Mus musculus molossinus, enhances meiotic recombination at a hotspot near the Lmp2 gene. Heterozygotes between the wm7 haplotype and the b or k haplotypes have yielded a high frequency of recombination (2.1%) in 1.3 kilobase kb segment of this hotspot. 20 refs., 2 figs.

  5. Cdc7-dependent phosphorylation of Mer2 facilitates initiation of yeast meiotic recombination.

    PubMed

    Sasanuma, Hiroyuki; Hirota, Kouji; Fukuda, Tomoyuki; Kakusho, Naoko; Kugou, Kazuto; Kawasaki, Yasuo; Shibata, Takehiko; Masai, Hisao; Ohta, Kunihiro

    2008-02-01

    Meiosis ensures genetic diversification of gametes and sexual reproduction. For successful meiosis, multiple events such as DNA replication, recombination, and chromosome segregation must occur coordinately in a strict regulated order. We investigated the meiotic roles of Cdc7 kinase in the initiation of meiotic recombination, namely, DNA double-strand breaks (DSBs) mediated by Spo11 and other coactivating proteins. Genetic analysis using bob1-1 cdc7Delta reveals that Cdc7 is essential for meiotic DSBs and meiosis I progression. We also demonstrate that the N-terminal region of Mer2, a Spo11 ancillary protein required for DSB formation and phosphorylated by cyclin-dependent kinase (CDK), contains two types of Cdc7-dependent phosphorylation sites near the CDK site (Ser30): One (Ser29) is essential for meiotic DSB formation, and the others exhibit a cumulative effect to facilitate DSB formation. Importantly, mutations on these sites confer severe defects in DSB formation even when the CDK phosphorylation is present at Ser30. Diploids of cdc7Delta display defects in the chromatin binding of not only Spo11 but also Rec114 and Mei4, other meiotic coactivators that may assist Spo11 binding to DSB hot spots. We thus propose that Cdc7, in concert with CDK, regulates Spo11 loading to DSB sites via Mer2 phosphorylation.

  6. Genome-Wide Association Study of Meiotic Recombination Phenotypes

    PubMed Central

    Begum, Ferdouse; Chowdhury, Reshmi; Cheung, Vivian G.; Sherman, Stephanie L.; Feingold, Eleanor

    2016-01-01

    Meiotic recombination is an essential step in gametogenesis, and is one that also generates genetic diversity. Genome-wide association studies (GWAS) and molecular studies have identified genes that influence of human meiotic recombination. RNF212 is associated with total or average number of recombination events, and PRDM9 is associated with the locations of hotspots, or sequences where crossing over appears to cluster. In addition, a common inversion on chromosome 17 is strongly associated with recombination. Other genes have been identified by GWAS, but those results have not been replicated. In this study, using new datasets, we characterized additional recombination phenotypes to uncover novel candidates and further dissect the role of already known loci. We used three datasets totaling 1562 two-generation families, including 3108 parents with 4304 children. We estimated five different recombination phenotypes including two novel phenotypes (average recombination counts within recombination hotspots and outside of hotspots) using dense SNP array genotype data. We then performed gender-specific and combined-sex genome-wide association studies (GWAS) meta-analyses. We replicated associations for several previously reported recombination genes, including RNF212 and PRDM9. By looking specifically at recombination events outside of hotspots, we showed for the first time that PRDM9 has different effects in males and females. We identified several new candidate loci, particularly for recombination events outside of hotspots. These include regions near the genes SPINK6, EVC2, ARHGAP25, and DLGAP2. This study expands our understanding of human meiotic recombination by characterizing additional features that vary across individuals, and identifying regulatory variants influencing the numbers and locations of recombination events. PMID:27733454

  7. Genome-Wide Association Study of Meiotic Recombination Phenotypes.

    PubMed

    Begum, Ferdouse; Chowdhury, Reshmi; Cheung, Vivian G; Sherman, Stephanie L; Feingold, Eleanor

    2016-12-07

    Meiotic recombination is an essential step in gametogenesis, and is one that also generates genetic diversity. Genome-wide association studies (GWAS) and molecular studies have identified genes that influence of human meiotic recombination. RNF212 is associated with total or average number of recombination events, and PRDM9 is associated with the locations of hotspots, or sequences where crossing over appears to cluster. In addition, a common inversion on chromosome 17 is strongly associated with recombination. Other genes have been identified by GWAS, but those results have not been replicated. In this study, using new datasets, we characterized additional recombination phenotypes to uncover novel candidates and further dissect the role of already known loci. We used three datasets totaling 1562 two-generation families, including 3108 parents with 4304 children. We estimated five different recombination phenotypes including two novel phenotypes (average recombination counts within recombination hotspots and outside of hotspots) using dense SNP array genotype data. We then performed gender-specific and combined-sex genome-wide association studies (GWAS) meta-analyses. We replicated associations for several previously reported recombination genes, including RNF212 and PRDM9 By looking specifically at recombination events outside of hotspots, we showed for the first time that PRDM9 has different effects in males and females. We identified several new candidate loci, particularly for recombination events outside of hotspots. These include regions near the genes SPINK6, EVC2, ARHGAP25, and DLGAP2 This study expands our understanding of human meiotic recombination by characterizing additional features that vary across individuals, and identifying regulatory variants influencing the numbers and locations of recombination events.

  8. DNA polymerase beta is critical for mouse meiotic synapsis.

    PubMed

    Kidane, Dawit; Jonason, Alan S; Gorton, Timothy S; Mihaylov, Ivailo; Pan, Jing; Keeney, Scott; de Rooij, Dirk G; Ashley, Terry; Keh, Agnes; Liu, Yanfeng; Banerjee, Urmi; Zelterman, Daniel; Sweasy, Joann B

    2010-01-20

    We have shown earlier that DNA polymerase beta (Pol beta) localizes to the synaptonemal complex (SC) during Prophase I of meiosis in mice. Pol beta localizes to synapsed axes during zygonema and pachynema, and it associates with the ends of bivalents during late pachynema and diplonema. To test whether these localization patterns reflect a function for Pol beta in recombination and/or synapsis, we used conditional gene targeting to delete the PolB gene from germ cells. We find that Pol beta-deficient spermatocytes are defective in meiotic chromosome synapsis and undergo apoptosis during Prophase I. We also find that SPO11-dependent gammaH2AX persists on meiotic chromatin, indicating that Pol beta is critical for the repair of SPO11-induced double-strand breaks (DSBs). Pol beta-deficient spermatocytes yielded reduced steady-state levels of the SPO11-oligonucleotide complexes that are formed when SPO11 is removed from the ends of DSBs, and cytological experiments revealed that chromosome-associated foci of replication protein A (RPA), RAD51 and DMC1 are less abundant in Pol beta-deficient spermatocyte nuclei. Localization of Pol beta to meiotic chromosomes requires the formation of SPO11-dependent DSBs. Taken together, these findings strongly indicate that Pol beta is required at a very early step in the processing of meiotic DSBs, at or before the removal of SPO11 from DSB ends and the generation of the 3' single-stranded tails necessary for subsequent strand exchange. The chromosome synapsis defects and Prophase I apoptosis of Pol beta-deficient spermatocytes are likely a direct consequence of these recombination defects.

  9. Transmission distortion affecting human noncrossover but not crossover recombination: a hidden source of meiotic drive.

    PubMed

    Odenthal-Hesse, Linda; Berg, Ingrid L; Veselis, Amelia; Jeffreys, Alec J; May, Celia A

    2014-02-01

    Meiotic recombination ensures the correct segregation of homologous chromosomes during gamete formation and contributes to DNA diversity through both large-scale reciprocal crossovers and very localised gene conversion events, also known as noncrossovers. Considerable progress has been made in understanding factors such as PRDM9 and SNP variants that influence the initiation of recombination at human hotspots but very little is known about factors acting downstream. To address this, we simultaneously analysed both types of recombinant molecule in sperm DNA at six highly active hotspots, and looked for disparity in the transmission of allelic variants indicative of any cis-acting influences. At two of the hotspots we identified a novel form of biased transmission that was exclusive to the noncrossover class of recombinant, and which presumably arises through differences between crossovers and noncrossovers in heteroduplex formation and biased mismatch repair. This form of biased gene conversion is not predicted to influence hotspot activity as previously noted for SNPs that affect recombination initiation, but does constitute a powerful and previously undetected source of recombination-driven meiotic drive that by extrapolation may affect thousands of recombination hotspots throughout the human genome. Intriguingly, at both of the hotspots described here, this drive favours strong (G/C) over weak (A/T) base pairs as might be predicted from the well-established correlations between high GC content and recombination activity in mammalian genomes.

  10. Meiotic recombination counteracts male-biased mutation (male-driven evolution)

    PubMed Central

    Mawaribuchi, Shuuji; Ito, Michihiko; Ogata, Mitsuaki; Oota, Hiroki; Katsumura, Takafumi; Takamatsu, Nobuhiko; Miura, Ikuo

    2016-01-01

    Meiotic recombination is believed to produce greater genetic variation despite the fact that deoxyribonucleic acid (DNA)-replication errors are a major source of mutations. In some vertebrates, mutation rates are higher in males than in females, which developed the theory of male-driven evolution (male-biased mutation). However, there is little molecular evidence regarding the relationships between meiotic recombination and male-biased mutation. Here we tested the theory using the frog Rana rugosa, which has both XX/XY- and ZZ/ZW-type sex-determining systems within the species. The male-to-female mutation-rate ratio (α) was calculated from homologous sequences on the X/Y or Z/W sex chromosomes, which supported male-driven evolution. Surprisingly, each α value was notably higher in the XX/XY-type group than in the ZZ/ZW-type group, although α should have similar values within a species. Interestingly, meiotic recombination between homologous chromosomes did not occur except at terminal regions in males of this species. Then, by subdividing α into two new factors, a replication-based male-to-female mutation-rate ratio (β) and a meiotic recombination-based XX-to-XY/ZZ-to-ZW mutation-rate ratio (γ), we constructed a formula describing the relationship among a nucleotide-substitution rate and the two factors, β and γ. Intriguingly, the β- and γ-values were larger and smaller than 1, respectively, indicating that meiotic recombination might reduce male-biased mutations. PMID:26791621

  11. Meiotic recombination counteracts male-biased mutation (male-driven evolution).

    PubMed

    Mawaribuchi, Shuuji; Ito, Michihiko; Ogata, Mitsuaki; Oota, Hiroki; Katsumura, Takafumi; Takamatsu, Nobuhiko; Miura, Ikuo

    2016-01-27

    Meiotic recombination is believed to produce greater genetic variation despite the fact that deoxyribonucleic acid (DNA)-replication errors are a major source of mutations. In some vertebrates, mutation rates are higher in males than in females, which developed the theory of male-driven evolution (male-biased mutation). However, there is little molecular evidence regarding the relationships between meiotic recombination and male-biased mutation. Here we tested the theory using the frog Rana rugosa, which has both XX/XY- and ZZ/ZW-type sex-determining systems within the species. The male-to-female mutation-rate ratio (α) was calculated from homologous sequences on the X/Y or Z/W sex chromosomes, which supported male-driven evolution. Surprisingly, each α value was notably higher in the XX/XY-type group than in the ZZ/ZW-type group, although α should have similar values within a species. Interestingly, meiotic recombination between homologous chromosomes did not occur except at terminal regions in males of this species. Then, by subdividing α into two new factors, a replication-based male-to-female mutation-rate ratio (β) and a meiotic recombination-based XX-to-XY/ZZ-to-ZW mutation-rate ratio (γ), we constructed a formula describing the relationship among a nucleotide-substitution rate and the two factors, β and γ. Intriguingly, the β- and γ-values were larger and smaller than 1, respectively, indicating that meiotic recombination might reduce male-biased mutations.

  12. The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

    PubMed Central

    Sekelsky, J. J.; McKim, K. S.; Chin, G. M.; Hawley, R. S.

    1995-01-01

    Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion. PMID:8647398

  13. A high throughput genetic screen identifies new early meiotic recombination functions in Arabidopsis thaliana.

    PubMed

    De Muyt, Arnaud; Pereira, Lucie; Vezon, Daniel; Chelysheva, Liudmila; Gendrot, Ghislaine; Chambon, Aurélie; Lainé-Choinard, Sandrine; Pelletier, Georges; Mercier, Raphaël; Nogué, Fabien; Grelon, Mathilde

    2009-09-01

    Meiotic recombination is initiated by the formation of numerous DNA double-strand breaks (DSBs) catalysed by the widely conserved Spo11 protein. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation; however, unlike Spo11, few of these are conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we took advantage of a high-throughput meiotic mutant screen carried out in the model plant Arabidopsis thaliana. A collection of 55,000 mutant lines was screened, and spo11-like mutations, characterised by a drastic decrease in chiasma formation at metaphase I associated with an absence of synapsis at prophase, were selected. This screen led to the identification of two populations of mutants classified according to their recombination defects: mutants that repair meiotic DSBs using the sister chromatid such as Atdmc1 or mutants that are unable to make DSBs like Atspo11-1. We found that in Arabidopsis thaliana at least four proteins are necessary for driving meiotic DSB repair via the homologous chromosomes. These include the previously characterised DMC1 and the Hop1-related ASY1 proteins, but also the meiotic specific cyclin SDS as well as the Hop2 Arabidopsis homologue AHP2. Analysing the mutants defective in DSB formation, we identified the previously characterised AtSPO11-1, AtSPO11-2, and AtPRD1 as well as two new genes, AtPRD2 and AtPRD3. Our data thus increase the number of proteins necessary for DSB formation in Arabidopsis thaliana to five. Unlike SPO11 and (to a minor extent) PRD1, these two new proteins are poorly conserved among species, suggesting that the DSB formation mechanism, but not its regulation, is conserved among eukaryotes.

  14. Both conserved and non-conserved regions of Spo11 are essential for meiotic recombination initiation in yeast.

    PubMed

    Nag, Dilip K; Pata, Janice D; Sironi, Manuela; Flood, David R; Hart, Ashley M

    2006-10-01

    DNA double-strand breaks (DSBs) are the initiators of most meiotic recombination events. In Saccharomyces cerevisiae, at least ten genes are necessary for meiotic DSB formation. However, the molecular roles of these proteins are not clearly understood. The meiosis-specific Spo11 protein, which shows sequence similarity with a subunit of an archaeal topoisomerase, is believed to catalyze the meiotic DSB formation. Spo11 is also required for induction of meiotic DSBs at long inverted repeats and at large trinucleotide repeat tracts. Here we report the isolation and characterization of temperature-sensitive spo11-mutant alleles to better understand how Spo11 functions, and how meiotic DSBs are generated at various recombination hotspots. Analysis of mutation sites of isolated spo11-mutant alleles indicated that both N-terminal and C-terminal non-conserved residues of Spo11 are essential for the protein's function, possibly for interaction with other meiotic DSB enzymes. Several of the mutation sites within the conserved region are predicted to lie on the surface of the protein, suggesting that this region is required for activation of the meiotic initiation complex via protein-protein interaction. In addition to the conditional mutants, we isolated partially recombination-defective mutants; analysis of one of these mutants indicated that Ski8, as observed previously, interacts with Spo11 via the latter's C-terminal residues.

  15. Control of meiotic recombination frequency in plant genomes.

    PubMed

    Henderson, Ian R

    2012-11-01

    Sexual eukaryotes reproduce via the meiotic cell division, where ploidy is halved and homologous chromosomes undergo reciprocal genetic exchange, termed crossover (CO). CO frequency has a profound effect on patterns of genetic variation and species evolution. Relative CO rates vary extensively both within and between plant genomes. Plant genome size varies by over 1000-fold, largely due to differential expansion of repetitive sequences, and increased genome size is associated with reduced CO frequency. Gene versus repeat sequences associate with distinct chromatin modifications, and evidence from plant genomes indicates that this epigenetic information influences CO patterns. This is consistent with data from diverse eukaryotes that demonstrate the importance of chromatin structure for control of meiotic recombination. In this review I will discuss CO frequency patterns in plant genomes and recent advances in understanding recombination distributions.

  16. Meiotic recombination analysis in female ducks (Anas platyrhynchos).

    PubMed

    Pigozzi, M I; Del Priore, L

    2016-06-01

    Meiotic recombination in female ducks was directly studied by immunolocalization of MLH1 protein, a mismatch repair protein of mature recombination nodules. In total, 6820 crossovers were scored along the autosomal synaptonemal complexes in 122 meiotic nuclei. From this analysis we predict that the female map length of the duck is 2845 cM, with a genome wide recombination rate of 2 cM/Mb. MLH1-focus mapping along the six largest bivalents shows regional variations of recombination frequencies that can be linked to differences in chromosome morphology. From this MLH1 mapping it can be inferred that distally located markers will appear more separated in genetic maps than physically equidistant markers located near the centromeres on bivalents 1 and 2. Instead, markers at interstitial positions on the acrocentric bivalents 3-6 will appear more tightly linked than expected on the basis of their physical distance because recombination is comparatively lower at the mid region of these chromosomes. The present results provide useful information to complement linkage mapping in ducks and extend previous knowledge about the variation of recombination rates among domestic Galloanserae.

  17. OsHUS1 facilitates accurate meiotic recombination in rice.

    PubMed

    Che, Lixiao; Wang, Kejian; Tang, Ding; Liu, Qiaoquan; Chen, Xiaojun; Li, Yafei; Hu, Qing; Shen, Yi; Yu, Hengxiu; Gu, Minghong; Cheng, Zhukuan

    2014-06-01

    Meiotic recombination normally takes place between allelic sequences on homologs. This process can also occur between non-allelic homologous sequences. Such ectopic interaction events can lead to chromosome rearrangements and are normally avoided. However, much remains unknown about how these ectopic interaction events are sensed and eliminated. In this study, using a screen in rice, we characterized a homolog of HUS1 and explored its function in meiotic recombination. In Oshus1 mutants, in conjunction with nearly normal homologous pairing and synapsis, vigorous, aberrant ectopic interactions occurred between nonhomologous chromosomes, leading to multivalent formation and subsequent chromosome fragmentation. These ectopic interactions relied on programmed meiotic double strand breaks and were formed in a manner independent of the OsMER3-mediated interference-sensitive crossover pathway. Although early homologous recombination events occurred normally, the number of interference-sensitive crossovers was reduced in the absence of OsHUS1. Together, our results indicate that OsHUS1 might be involved in regulating ectopic interactions during meiosis, probably by forming the canonical RAD9-RAD1-HUS1 (9-1-1) complex.

  18. Factors influencing recombination frequency and distribution in a human meiotic crossover hotspot.

    PubMed

    Jeffreys, Alec J; Neumann, Rita

    2005-08-01

    Little is known about the factors that influence the frequency and distribution of meiotic recombination events within human crossover hotspots. We now describe the detailed analysis of sperm recombination in the NID1 hotspot. Like the neighbouring MS32 hotspot, the NID1 hotspot is associated with a minisatellite, suggesting that hotspots predispose DNA to tandem repetition. Unlike MS32, crossover resolution breakpoints in NID1 avoid the minisatellite, producing a cold spot within the hotspot. This avoidance may be related to the palindromic nature of the minisatellite interfering with the generation and/or processing of recombination intermediates. The NID1 hotspot also contains a single nucleotide polymorphism (SNP) close to the centre, which appears to directly influence the frequency of crossover initiation. Quantitative gene conversion assays show that this SNP affects the frequency of gene conversion and crossover to a very similar extent, providing evidence that conversions and crossovers are triggered by the same recombination initiating events. The recombination-suppressing allele is over-transmitted to recombinant progeny, and provides the most dramatic example to date of recombination-mediated meiotic drive, of a magnitude sufficient to virtually guarantee that the recombination suppressor will eventually replace the more active allele in human populations.

  19. ARG-walker: inference of individual specific strengths of meiotic recombination hotspots by population genomics analysis

    PubMed Central

    2015-01-01

    Background Meiotic recombination hotspots play important roles in various aspects of genomics, but the underlying mechanisms for regulating the locations and strengths of recombination hotspots are not yet fully revealed. Most existing algorithms for estimating recombination rates from sequence polymorphism data can only output average recombination rates of a population, although there is evidence for the heterogeneity in recombination rates among individuals. For genome-wide association studies (GWAS) of recombination hotspots, an efficient algorithm that estimates the individualized strengths of recombination hotspots is highly desirable. Results In this work, we propose a novel graph mining algorithm named ARG-walker, based on random walks on ancestral recombination graphs (ARG), to estimate individual-specific recombination hotspot strengths. Extensive simulations demonstrate that ARG-walker is able to distinguish the hot allele of a recombination hotspot from the cold allele. Integrated with output of ARG-walker, we performed GWAS on the phased haplotype data of the 22 autosome chromosomes of the HapMap Asian population samples of Chinese and Japanese (JPT+CHB). Significant cis-regulatory signals have been detected, which is corroborated by the enrichment of the well-known 13-mer motif CCNCCNTNNCCNC of PRDM9 protein. Moreover, two new DNA motifs have been identified in the flanking regions of the significantly associated SNPs (single nucleotide polymorphisms), which are likely to be new cis-regulatory elements of meiotic recombination hotspots of the human genome. Conclusions Our results on both simulated and real data suggest that ARG-walker is a promising new method for estimating the individual recombination variations. In the future, it could be used to uncover the mechanisms of recombination regulation and human diseases related with recombination hotspots. PMID:26679564

  20. Functional Roles of Acetylated Histone Marks at Mouse Meiotic Recombination Hot Spots.

    PubMed

    Getun, Irina V; Wu, Zhen; Fallahi, Mohammad; Ouizem, Souad; Liu, Qin; Li, Weimin; Costi, Roberta; Roush, William R; Cleveland, John L; Bois, Philippe R J

    2017-02-01

    Meiotic recombination initiates following the formation of DNA double-strand breaks (DSBs) by the Spo11 endonuclease early in prophase I, at discrete regions in the genome coined "hot spots." In mammals, meiotic DSB site selection is directed in part by sequence-specific binding of PRDM9, a polymorphic histone H3 (H3K4Me3) methyltransferase. However, other chromatin features needed for meiotic hot spot specification are largely unknown. Here we show that the recombinogenic cores of active hot spots in mice harbor several histone H3 and H4 acetylation and methylation marks that are typical of open, active chromatin. Further, deposition of these open chromatin-associated histone marks is dynamic and is manifest at spermatogonia and/or pre-leptotene-stage cells, which facilitates PRDM9 binding and access for Spo11 to direct the formation of DSBs, which are initiated at the leptotene stage. Importantly, manipulating histone acetylase and deacetylase activities established that histone acetylation marks are necessary for both hot spot activity and crossover resolution. We conclude that there are functional roles for histone acetylation marks at mammalian meiotic recombination hot spots.

  1. Understanding and Manipulating Meiotic Recombination in Plants[OPEN

    PubMed Central

    2017-01-01

    Meiosis is a specialized cell division, essential in most reproducing organisms to halve the number of chromosomes, thereby enabling the restoration of ploidy levels during fertilization. A key step of meiosis is homologous recombination, which promotes homologous pairing and generates crossovers (COs) to connect homologous chromosomes until their separation at anaphase I. These CO sites, seen cytologically as chiasmata, represent a reciprocal exchange of genetic information between two homologous nonsister chromatids. This gene reshuffling during meiosis has a significant influence on evolution and also plays an essential role in plant breeding, because a successful breeding program depends on the ability to bring the desired combinations of alleles on chromosomes. However, the number and distribution of COs during meiosis is highly constrained. There is at least one CO per chromosome pair to ensure accurate segregation of homologs, but in most organisms, the CO number rarely exceeds three regardless of chromosome size. Moreover, their positions are not random on chromosomes but exhibit regional preference. Thus, genes in recombination-poor regions tend to be inherited together, hindering the generation of novel allelic combinations that could be exploited by breeding programs. Recently, much progress has been made in understanding meiotic recombination. In particular, many genes involved in the process in Arabidopsis (Arabidopsis thaliana) have been identified and analyzed. With the coming challenges of food security and climate change, and our enhanced knowledge of how COs are formed, the interest and needs in manipulating CO formation are greater than ever before. In this review, we focus on advances in understanding meiotic recombination and then summarize the attempts to manipulate CO formation. Last, we pay special attention to the meiotic recombination in polyploidy, which is a common genomic feature for many crop plants. PMID:28108697

  2. Have a break: determinants of meiotic DNA double strand break (DSB) formation and processing in plants.

    PubMed

    Edlinger, Bernd; Schlögelhofer, Peter

    2011-03-01

    Meiosis is an essential process for sexually reproducing organisms, leading to the formation of specialized generative cells. This review intends to highlight current knowledge of early events during meiosis derived from various model organisms, including plants. It will particularly focus on cis- and trans-requirements of meiotic DNA double strand break (DSB) formation, a hallmark event during meiosis and a prerequisite for recombination of genetic traits. Proteins involved in DSB formation in different organisms, emphasizing the known factors from plants, will be introduced and their functions outlined. Recent technical advances in DSB detection and meiotic recombination analysis will be reviewed, as these new tools now allow analysis of early meiotic recombination in plants with incredible accuracy. To anticipate future directions in plant meiosis research, unpublished results will be included wherever possible.

  3. The MRE4 gene encodes a novel protein kinase homologue required for meiotic recombination in Saccharomyces cerevisiae.

    PubMed Central

    Leem, S H; Ogawa, H

    1992-01-01

    The MRE4 gene was cloned by complementation of the defects of meiotic recombination and haploidization in an mre4-1 mutant. Disruption of MRE4 resulted in reduced meiotic recombination and spore inviability. The mre4 spore lethality can be suppressed by spo13, a mutation that causes cells to bypass the reductional division. Analysis of meiotic DNA extracted from the mre4 mutant cells revealed that double-strand breaks occurred at the two sites of the HIS4-LEU2 recombination hot spot, but at a frequency of about 10-20% of the wild type. Northern blot analysis indicated that the MRE4 gene produces four transcripts of 1.63, 3.2, 4.0 and 6.2 kb. All of these transcripts are absent from mitotic cells and are meiotically induced. The DNA sequence of the MRE4 open reading frame predicts a 497-amino acids protein with a molecular mass of 56.8 kDa. The Mre4 protein contains highly conserved amino acid sequences found specifically in serine-threonine protein kinases. These results suggest that protein phosphorylation is required directly or indirectly for meiotic recombination. Images PMID:1741279

  4. Arabidopsis SPO11-2 functions with SPO11-1 in meiotic recombination.

    PubMed

    Stacey, Nicola J; Kuromori, Takashi; Azumi, Yoshitaka; Roberts, Gethin; Breuer, Christian; Wada, Takuji; Maxwell, Anthony; Roberts, Keith; Sugimoto-Shirasu, Keiko

    2006-10-01

    The Spo11 protein is a eukaryotic homologue of the archaeal DNA topoisomerase VIA subunit (topo VIA). In archaea it is involved, together with its B subunit (topo VIB), in DNA replication. However, most eukaryotes, including yeasts, insects and vertebrates, instead have a single gene for Spo11/topo VIA and no homologues for topo VIB. In these organisms, Spo11 mediates DNA double-strand breaks that initiate meiotic recombination. Many plant species, in contrast to other eukaryotes, have three homologues for Spo11/topo VIA and one for topo VIB. The homologues in Arabidopsis, AtSPO11-1, AtSPO11-2 and AtSPO11-3, all share 20-30% sequence similarity with other Spo11/topo VIA proteins, but their functional relationship during meiosis or other processes is not well understood. Previous genetic evidence suggests that AtSPO11-1 is a true orthologue of Spo11 in other eukaryotes and is required for meiotic recombination, whereas AtSPO11-3 is involved in DNA endo-reduplication as a part of the topo VI complex. In this study, we show that plants homozygous for atspo11-2 exhibit a severe sterility phenotype. Both male and female meiosis are severely disrupted in the atspo11-2 mutant, and this is associated with severe defects in synapsis during the first meiotic division and reduced meiotic recombination. Further genetic analysis revealed that AtSPO11-1 and AtSPO11-2 genetically interact, i.e. plants heterozygous for both atspo11-1 and atspo11-2 are also sterile, suggesting that AtSPO11-1 and AtSPO11-2 have largely overlapping functions. Thus, the three Arabidopsis Spo11 homologues appear to function in two discrete processes, i.e. AtSPO11-1 and AtSPO11-2 in meiotic recombination and AtSPO11-3 in DNA replication.

  5. Sister cohesion and structural axis components mediate homolog bias of meiotic recombination

    PubMed Central

    Kim, Keun P.; Weiner, Beth M.; Zhang, Liangran; Jordan, Amy; Dekker, Job; Kleckner, Nancy

    2010-01-01

    SUMMARY Meiotic recombination occurs between one chromatid of each maternal and paternal homolog (homolog bias) versus between sister chromatids (sister bias). Physical DNA analysis reveals that meiotic cohesin/axis component Rec8 promotes sister bias, likely via its cohesion activity. Two meiosis-specific axis components, Red1/Mek1kinase, counteract this effect. With this precondition satisfied, other molecules directly specify homolog bias per se. Rec8 also acts positively to maintain homolog bias during crossover recombination. These observations point to sequential release of double-strand break ends from association with their sister. Red1 and Rec8 are found to play distinct roles for sister cohesion, DSB formation and recombination progression kinetics. Also, the two components are enriched in spatially distinct domains of axial structure that develop prior to DSB formation. We propose that Red1 and Rec8 domains provide functionally complementary environments whereby inputs evolved from DSB repair and late-stage chromosome morphogenesis are integrated to give the complete meiotic chromosomal program. PMID:21145459

  6. Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion

    PubMed Central

    Laureau, Raphaëlle; Loeillet, Sophie; Salinas, Francisco; Bergström, Anders; Legoix-Né, Patricia; Liti, Gianni; Nicolas, Alain

    2016-01-01

    In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction. PMID:26828862

  7. New paradigms for conserved, multifactorial, cis-acting regulation of meiotic recombination

    PubMed Central

    Wahls, Wayne P.; Davidson, Mari K.

    2012-01-01

    How do cells position the Spo11 (Rec12)-dependent initiation of meiotic recombination at hotspots? The mechanisms are poorly understood and a prevailing view is that they differ substantially between phylogenetic groups. However, recent work discovered that individual species have multiple different DNA sequence-specific, protein–DNA complexes that regulate (and are essential for the activation of) recombination hotspots. The cis-acting elements function combinatorially with documented examples of synergism, antagonism and redundancy. Furthermore, we provide evidence that all currently well-defined modules of this multifactorial, cis-acting regulation are conserved functionally between taxa whose latest common ancestor occurred more than 1 billion years ago. Functionally conserved components include the ATF/CREB-family heterodimer Atf1-Pcr1 and its CRE-like DNA site M26, the CCAAT-box-binding complex Php2-Php3-Php5 and the CCAAT-box, and the zinc-finger protein Rst2 and its Oligo-C motif. The newfound multiplicity, functional redundancy and conservation of cis-acting controls constitute a paradigm shift with broad implications. They provide compelling evidence that most meiotic recombination is, like transcription, regulated by sequence-specific protein–DNA complexes. And the new findings provide important mechanistic insight, such as a solution to the conundrum that Prdm9 is a ‘master regulator’ of—yet is dispensable for—hotspot activity in mammals. PMID:22904082

  8. Processing of meiotic DNA double strand breaks requires cyclin-dependent kinase and multiple nucleases.

    PubMed

    Manfrini, Nicola; Guerini, Ilaria; Citterio, Andrea; Lucchini, Giovanna; Longhese, Maria Pia

    2010-04-09

    Meiotic recombination requires the formation of programmed Spo11-dependent DNA double strand breaks (DSBs). In Saccharomyces cerevisiae, the Sae2 protein and the Mre11-Rad50-Xrs2 complex are necessary to remove the covalently attached Spo11 protein from the DNA ends, which are then resected by so far unknown nucleases. Here, we demonstrate that phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends. This finding suggests that Cdk1 activity is required for the processing of Spo11-induced DSBs, thus providing a mechanism for coordinating DSB resection with progression through meiotic prophase. Furthermore, the helicase Sgs1 and the nucleases Exo1 and Dna2 participate in lengthening the 5'-3' resection tracts during meiosis by controlling a step subsequent to Spo11 removal.

  9. The TopoVIB-Like protein family is required for meiotic DNA double-strand break formation.

    PubMed

    Robert, T; Nore, A; Brun, C; Maffre, C; Crimi, B; Bourbon, H-M; de Massy, B

    2016-02-26

    Meiotic recombination is induced by the formation of DNA double-strand breaks (DSBs) catalyzed by SPO11, the ortholog of subunit A of TopoVI DNA topoisomerase (TopoVIA). TopoVI activity requires the interaction between A and B subunits. We identified a conserved family of plant and animal proteins [the TOPOVIB-Like (TOPOVIBL) family] that share strong structural similarity to the TopoVIB subunit of TopoVI DNA topoisomerase. We further characterize the meiotic recombination proteins Rec102 (Saccharomyces cerevisiae), Rec6 (Schizosaccharomyces pombe), and MEI-P22 (Drosophila melanogaster) as homologs to the transducer domain of TopoVIB. We demonstrate that the mouse TOPOVIBL protein interacts and forms a complex with SPO11 and is required for meiotic DSB formation. We conclude that meiotic DSBs are catalyzed by a complex involving SPO11 and TOPOVIBL.

  10. Enrichment of meiotic recombination hotspot sequences by avidin capture technology2

    PubMed Central

    Teixeira, Daniel Camara; Malkaram, Sridhar A.

    2013-01-01

    About 40% of the hotspots for meiotic recombination contain the degenerate consensus sequence 5’-CCNCCNTNNCCNC-3’. Here we present a novel protocol for enriching hotspot sequences from digested genomic DNA by using biotinylated oligonucleotides and streptavidin-coated magnetic beads. The captured hotspots can be released by simple digestion with restriction enzymes for subsequent characterization by second generation sequencing or PCR. The capture protocol specifically enriches hotspot sequences, judged by using fluorophore-conjugated synthetic oligonucleotides and synthetic double-stranded oligonucleotides in combination with PCR. The capture protocol enriches single stranded DNA, denatured double-stranded DNA, and large fragments (>3,000 bp) of digested plasmid DNA with good efficacy. No false positive and false negatives were detected when enriching digested DNA from human cell cultures and primary human cells. The protocol can probably be adapted to enriching sequences other than the hotspot sequence by altering the sequence in the capture oligonucleotide. We intend to apply this protocol in studies assessing effects of micronutrient status on meiotic recombination events in human sperm. PMID:23270922

  11. Mek1/Mre4 is a master regulator of meiotic recombination in budding yeast

    PubMed Central

    Hollingsworth, Nancy M.

    2016-01-01

    Sexually reproducing organisms create gametes with half the somatic cell chromosome number so that fusion of gametes at fertilization does not change the ploidy of the cell. This reduction in chromosome number occurs by the specialized cell division of meiosis in which two rounds of chromosome segregation follow a single round of chromosome duplication. Meiotic crossovers formed between the non-sister chromatids of homologous chromosomes, combined with sister chromatid cohesion, physically connect homologs, thereby allowing proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) whose repair is highly regulated such that (1) there is a bias for recombination with homologs rather than sister chromatids, (2) crossovers are distributed throughout the genome by a process called interference, (3) crossover homeostasis regulates the balance between crossover and non-crossover repair to maintain a critical number of crossovers and (4) each pair of homologs receives at least one crossover. It was previously known that the imposition of interhomolog bias in budding yeast requires meiosis-specific modifications to the DNA damage response and the local activation of the meiosis-specific Mek1/Mre4 (hereafter Mek1) kinase at DSBs. However, because inactivation of Mek1 results in intersister, rather than interhomolog DSB repair, whether Mek1 had a role in interhomolog pathway choice was unknown. A recent study by Chen et al. (2015) reveals that Mek1 indirectly regulates the crossover/non-crossover decision between homologs as well as genetic interference. It does this by enabling phosphorylation of Zip1, the meiosis-specific transverse filament protein of the synaptonemal complex (SC), by the conserved cell cycle kinase, Cdc7-Dbf4 (DDK). These results suggest that Mek1 is a “master regulator” of meiotic recombination in budding yeast.

  12. Meiotic Recombination Hotspots of Fission Yeast Are Directed to Loci that Express Non-Coding RNA

    PubMed Central

    Wahls, Wayne P.; Siegel, Eric R.; Davidson, Mari K.

    2008-01-01

    Background Polyadenylated, mRNA-like transcripts with no coding potential are abundant in eukaryotes, but the functions of these long non-coding RNAs (ncRNAs) are enigmatic. In meiosis, Rec12 (Spo11) catalyzes the formation of dsDNA breaks (DSBs) that initiate homologous recombination. Most meiotic recombination is positioned at hotspots, but knowledge of the mechanisms is nebulous. In the fission yeast genome DSBs are located within 194 prominent peaks separated on average by 65-kbp intervals of DNA that are largely free of DSBs. Methodology/Principal Findings We compared the genome-wide distribution of DSB peaks to that of polyadenylated ncRNA molecules of the prl class. DSB peaks map to ncRNA loci that may be situated within ORFs, near the boundaries of ORFs and intergenic regions, or most often within intergenic regions. Unconditional statistical tests revealed that this colocalization is non-random and robust (P≤5.5×10−8). Furthermore, we tested and rejected the hypothesis that the ncRNA loci and DSB peaks localize preferentially, but independently, to a third entity on the chromosomes. Conclusions/Significance Meiotic DSB hotspots are directed to loci that express polyadenylated ncRNAs. This reveals an unexpected, possibly unitary mechanism for what directs meiotic recombination to hotspots. It also reveals a likely biological function for enigmatic ncRNAs. We propose specific mechanisms by which ncRNA molecules, or some aspect of RNA metabolism associated with ncRNA loci, help to position recombination protein complexes at DSB hotspots within chromosomes. PMID:18682829

  13. Self-Organization of Meiotic Recombination Initiation: General Principles and Molecular Pathways

    PubMed Central

    Keeney, Scott; Lange, Julian; Mohibullah, Neeman

    2015-01-01

    Recombination in meiosis is a fascinating case study for the coordination of chromosomal duplication, repair, and segregation with each other and with progression through a cell-division cycle. Meiotic recombination initiates with formation of developmentally programmed DNA double-strand breaks (DSBs) at many places across the genome. DSBs are important for successful meiosis but are also dangerous lesions that can mutate or kill, so cells ensure that DSBs are made only at the right times, places, and amounts. This review examines the complex web of pathways that accomplish this control. We explore how chromosome breakage is integrated with meiotic progression and how feedback mechanisms spatially pattern DSB formation and make it homeostatic, robust, and error-correcting. Common regulatory themes recur in different organisms or in different contexts in the same organism. We review this evolutionary and mechanistic conservation but also highlight where control modules have diverged. The framework that emerges helps explain how meiotic chromosomes behave as a self-organizing system. PMID:25421598

  14. A Discrete Class of Intergenic DNA Dictates Meiotic DNA Break Hotspots in Fission Yeast

    PubMed Central

    Cam, Hugh P; Farah, Joseph A; Grewal, Shiv I. S; Smith, Gerald R

    2007-01-01

    Meiotic recombination is initiated by DNA double-strand breaks (DSBs) made by Spo11 (Rec12 in fission yeast), which becomes covalently linked to the DSB ends. Like recombination events, DSBs occur at hotspots in the genome, but the genetic factors responsible for most hotspots have remained elusive. Here we describe in fission yeast the genome-wide distribution of meiosis-specific Rec12-DNA linkages, which closely parallel DSBs measured by conventional Southern blot hybridization. Prominent DSB hotspots are located ∼65 kb apart, separated by intervals with little or no detectable breakage. Most hotspots lie within exceptionally large intergenic regions. Thus, the chromosomal architecture responsible for hotspots in fission yeast is markedly different from that of budding yeast, in which DSB hotspots are much more closely spaced and, in many regions of the genome, occur at each promoter. Our analysis in fission yeast reveals a clearly identifiable chromosomal feature that can predict the majority of recombination hotspots across a whole genome and provides a basis for searching for the chromosomal features that dictate hotspots of meiotic recombination in other organisms, including humans. PMID:17722984

  15. Extensive Interallelic Polymorphisms Drive Meiotic Recombination into a Crossover Pathway

    PubMed Central

    Dooner, Hugo K.

    2002-01-01

    Recombinants isolated from most meiotic intragenic recombination experiments in maize, but not in yeast, are borne principally on crossover chromosomes. This excess of crossovers is not explained readily by the canonical double-strand break repair model of recombination, proposed to account for a large body of yeast data, which predicts that crossovers (COs) and noncrossovers (NCOs) should be recovered equally. An attempt has been made here to identify general rules governing the recovery of the CO and NCO classes of intragenic recombinants in maize. Recombination was analyzed in bz heterozygotes between a variety of mutations derived from the same or different progenitor alleles. The mutations include point mutations, transposon insertions, and transposon excision footprints. Consequently, the differences between the bz heteroalleles ranged from just two nucleotides to many nucleotides, indels, and insertions. In this article, allelic pairs differing at only two positions are referred to as dimorphic to distinguish them from polymorphic pairs, which differ at multiple positions. The present study has revealed the following effects at these bz heteroalleles: (1) recombination between polymorphic heteroalleles produces mostly CO chromosomes; (2) recombination between dimorphic heteroalleles produces both CO and NCO chromosomes, in ratios apparently dependent on the nature of the heteroalleles; and (3) in dimorphic heterozygotes, the two NCO classes are recovered in approximately equal numbers when the two mutations are point mutations but not when one or both mutations are insertions. These observations are discussed in light of a recent version of the double-strand break repair model of recombination that postulates separate pathways for the formation of CO and NCO products. PMID:12034905

  16. PHS1 regulates meiotic recombination and homologous chromosome pairing by controlling the transport of RAD50 to the nucleus.

    PubMed

    Ronceret, Arnaud; Doutriaux, Marie-Pascale; Golubovskaya, Inna N; Pawlowski, Wojciech P

    2009-11-24

    Recombination and pairing of homologous chromosomes are critical for bivalent formation in meiotic prophase. In many organisms, including yeast, mammals, and plants, pairing and recombination are intimately interconnected. The POOR HOMOLOGOUS SYNAPSIS1 (PHS1) gene acts in coordination of chromosome pairing and early recombination steps in plants, ensuring pairing fidelity and proper repair of meiotic DNA double-strand-breaks. In phs1 mutants, chromosomes exhibit early recombination defects and frequently associate with non-homologous partners, instead of pairing with their proper homologs. Here, we show that the product of the PHS1 gene is a cytoplasmic protein that functions by controlling transport of RAD50 from cytoplasm to the nucleus. RAD50 is a component of the MRN protein complex that processes meiotic double-strand-breaks to produce single-stranded DNA ends, which act in the homology search and recombination. We demonstrate that PHS1 plays the same role in homologous pairing in both Arabidopsis and maize, whose genomes differ dramatically in size and repetitive element content. This suggests that PHS1 affects pairing of the gene-rich fraction of the genome rather than preventing pairing between repetitive DNA elements. We propose that PHS1 is part of a system that regulates the progression of meiotic prophase by controlling entry of meiotic proteins into the nucleus. We also document that in phs1 mutants in Arabidopsis, centromeres interact before pairing commences along chromosome arms. Centromere coupling was previously observed in yeast and polyploid wheat while our data suggest that it may be a more common feature of meiosis.

  17. Identification of New Genes Required for Meiotic Recombination in Saccharomyces Cerevisiae

    PubMed Central

    Ajimura, M.; Leem, S. H.; Ogawa, H.

    1993-01-01

    Mutants defective in meiotic recombination were isolated from a disomic haploid strain of Saccharomyces cerevisiae by examining recombination within the leu2 and his4 heteroalleles located on chromosome III. The mutants were classified into two new complementation groups (MRE2 and MRE11) and eight previously identified groups, which include SPO11, HOP1, REC114, MRE4/MEK1 and genes in the RAD52 epistasis group. All of the mutants, in which the mutations in the new complementation groups are homozygous and diploid, can undergo premeiotic DNA synthesis and produce spores. The spores are, however, not viable. The mre2 and mre11 mutants produce viable spores in a spo13 background, in which meiosis I is bypassed, suggesting that these mutants are blocked at an early step in meiotic recombination. The mre2 mutant does not exhibit any unusual phenotype during mitosis and it is, thus, considered to have a mutation in a meiosis-specific gene. By contrast, the mre11 mutant is sensitive to damage to DNA by methyl methanesulfonate and exhibits a hyperrecombination phenotype in mitosis. Among six alleles of HOP1 that were isolated, an unusual pattern of intragenic complementation was observed. PMID:8417989

  18. CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination.

    PubMed

    Marjanović, Marko; Sánchez-Huertas, Carlos; Terré, Berta; Gómez, Rocío; Scheel, Jan Frederik; Pacheco, Sarai; Knobel, Philip A; Martínez-Marchal, Ana; Aivio, Suvi; Palenzuela, Lluís; Wolfrum, Uwe; McKinnon, Peter J; Suja, José A; Roig, Ignasi; Costanzo, Vincenzo; Lüders, Jens; Stracker, Travis H

    2015-07-09

    CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63-deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death, and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63-deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination.

  19. Meiotic Recombination Initiation in and around Retrotransposable Elements in Saccharomyces cerevisiae

    PubMed Central

    Sasaki, Mariko; Keeney, Scott

    2013-01-01

    Meiotic recombination is initiated by large numbers of developmentally programmed DNA double-strand breaks (DSBs), ranging from dozens to hundreds per cell depending on the organism. DSBs formed in single-copy sequences provoke recombination between allelic positions on homologous chromosomes, but DSBs can also form in and near repetitive elements such as retrotransposons. When they do, they create a risk for deleterious genome rearrangements in the germ line via recombination between non-allelic repeats. A prior study in budding yeast demonstrated that insertion of a Ty retrotransposon into a DSB hotspot can suppress meiotic break formation, but properties of Ty elements in their most common physiological contexts have not been addressed. Here we compile a comprehensive, high resolution map of all Ty elements in the rapidly and efficiently sporulating S. cerevisiae strain SK1 and examine DSB formation in and near these endogenous retrotransposable elements. SK1 has 30 Tys, all but one distinct from the 50 Tys in S288C, the source strain for the yeast reference genome. From whole-genome DSB maps and direct molecular assays, we find that DSB levels and chromatin structure within and near Tys vary widely between different elements and that local DSB suppression is not a universal feature of Ty presence. Surprisingly, deletion of two Ty elements weakened adjacent DSB hotspots, revealing that at least some Ty insertions promote rather than suppress nearby DSB formation. Given high strain-to-strain variability in Ty location and the high aggregate burden of Ty-proximal DSBs, we propose that meiotic recombination is an important component of host-Ty interactions and that Tys play critical roles in genome instability and evolution in both inbred and outcrossed sexual cycles. PMID:24009525

  20. The mismatch repair system reduces meiotic homeologous recombination and stimulates recombination-dependent chromosome loss.

    PubMed Central

    Chambers, S R; Hunter, N; Louis, E J; Borts, R H

    1996-01-01

    Efficient genetic recombination requires near-perfect homology between participating molecules. Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system. The effects of chromosomal divergence in diploids of Saccharomyces cerevisiae in which one copy of chromosome III is derived from a closely related species, Saccharomyces paradoxus, have been examined. Meiotic recombination between the diverged chromosomes is decreased by 25-fold. Spore viability is reduced with an observable increase in the number of tetrads with only two or three viable spores. Asci with only two viable spores are disomic for chromosome III, consistent with meiosis I nondisjunction of the homeologs. Asci with three viable spores are highly enriched for recombinants relative to tetrads with four viable spores. In 96% of the class with three viable spores, only one spore possesses a recombinant chromosome III, suggesting that the recombination process itself contributes to meiotic death. This phenomenon is dependent on the activities of the mismatch repair genes PMS1 and MSH2. A model of mismatch-stimulated chromosome loss is proposed to account for this observation. As expected, crossing over is increased in pms1 and msh2 mutants. Furthermore, genetic exchange in pms1 msh2 double mutants is affected to a greater extent than in either mutant alone, suggesting that the two proteins act independently to inhibit homeologous recombination. All mismatch repair-deficient strains exhibited reductions in the rate of chromosome III nondisjunction. PMID:8887641

  1. X chromosome effect on maternal recombination and meiotic drive in the mouse.

    PubMed Central

    de La Casa-Esperón, Elena; Loredo-Osti, J Concepción; Pardo-Manuel de Villena, Fernando; Briscoe, Tammi L; Malette, Jan Michel; Vaughan, Joe E; Morgan, Kenneth; Sapienza, Carmen

    2002-01-01

    We observed that maternal meiotic drive favoring the inheritance of DDK alleles at the Om locus on mouse chromosome 11 was correlated with the X chromosome inactivation phenotype of (C57BL/6-Pgk1(a) x DDK)F(1) mothers. The basis for this unexpected observation appears to lie in the well-documented effect of recombination on meiotic drive that results from nonrandom segregation of chromosomes. Our analysis of genome-wide levels of meiotic recombination in females that vary in their X-inactivation phenotype indicates that an allelic difference at an X-linked locus is responsible for modulating levels of recombination in oocytes. PMID:12196408

  2. DNase I-hypersensitive sites and transcription factor-binding motifs within the mouse E beta meiotic recombination hot spot.

    PubMed

    Shenkar, R; Shen, M H; Arnheim, N

    1991-04-01

    The second intron of the E beta gene in the mouse major histocompatibility complex is the site of a meiotic recombination hot spot. We detected two DNase I-hypersensitive sites in this intron in meiotic cells isolated from mouse testes. One site appears to be constitutive and is found in other tissues regardless of whether or not they express the E beta gene. Near this hypersensitive site are potential binding motifs for H2TF1/KBF1, NF kappa B, and octamer transcription factors. Gel retardation studies with mouse lymphoma cell nuclear extracts confirmed that each of these motifs is capable of binding protein. The binding of transcription factors may contribute to the enhancement of recombination potential by altering chromatin structure and increasing the accessibility of the DNA to the recombination machinery.

  3. Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination.

    PubMed

    Panizza, Silvia; Mendoza, Marco A; Berlinger, Marc; Huang, Lingzhi; Nicolas, Alain; Shirahige, Katsuhiko; Klein, Franz

    2011-08-05

    Meiotic recombination between homologous chromosomes initiates via programmed DNA double-strand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2, and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in a domain-specific fashion by cohesin. Loss of Rec114, Mer2, and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.

  4. Identification of DSB-1, a protein required for initiation of meiotic recombination in Caenorhabditis elegans, illuminates a crossover assurance checkpoint.

    PubMed

    Stamper, Ericca L; Rodenbusch, Stacia E; Rosu, Simona; Ahringer, Julie; Villeneuve, Anne M; Dernburg, Abby F

    2013-01-01

    Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs). DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene, dsb-1, that is specifically required for meiotic DSB formation in the nematode Caenorhabditis elegans. DSB-1 localizes to chromosomes during early meiotic prophase, coincident with the timing of DSB formation. DSB-1 also promotes normal protein levels and chromosome localization of DSB-2, a paralogous protein that plays a related role in initiating recombination. Mutations that disrupt crossover formation result in prolonged DSB-1 association with chromosomes, suggesting that nuclei may remain in a DSB-permissive state. Extended DSB-1 localization is seen even in mutants with defects in early recombination steps, including spo-11, suggesting that the absence of crossover precursors triggers the extension. Strikingly, failure to form a crossover precursor on a single chromosome pair is sufficient to extend the localization of DSB-1 on all chromosomes in the same nucleus. Based on these observations we propose a model for crossover assurance that acts through DSB-1 to maintain a DSB-permissive state until all chromosome pairs acquire crossover precursors. This work identifies a novel component of the DSB machinery in C. elegans, and sheds light on an important pathway that regulates DSB formation for crossover assurance.

  5. MEI4 – a central player in the regulation of meiotic DNA double-strand break formation in the mouse.

    PubMed

    Kumar, Rajeev; Ghyselinck, Norbert; Ishiguro, Kei-ichiro; Watanabe, Yoshinori; Kouznetsova, Anna; Höög, Christer; Strong, Edward; Schimenti, John; Daniel, Katrin; Toth, Attila; de Massy, Bernard

    2015-05-01

    The formation of programmed DNA double-strand breaks (DSBs) at the beginning of meiotic prophase marks the initiation of meiotic recombination. Meiotic DSB formation is catalyzed by SPO11 and their repair takes place on meiotic chromosome axes. The evolutionarily conserved MEI4 protein is required for meiotic DSB formation and is localized on chromosome axes. Here, we show that HORMAD1, one of the meiotic chromosome axis components, is required for MEI4 localization. Importantly, the quantitative correlation between the level of axis-associated MEI4 and DSB formation suggests that axis-associated MEI4 could be a limiting factor for DSB formation. We also show that MEI1, REC8 and RAD21L are important for proper MEI4 localization. These findings on MEI4 dynamics during meiotic prophase suggest that the association of MEI4 to chromosome axes is required for DSB formation, and that the loss of this association upon DSB repair could contribute to turning off meiotic DSB formation.

  6. Dbl2 Regulates Rad51 and DNA Joint Molecule Metabolism to Ensure Proper Meiotic Chromosome Segregation

    PubMed Central

    Hyppa, Randy W.; Benko, Zsigmond; Misova, Ivana; Schleiffer, Alexander; Smith, Gerald R.; Gregan, Juraj

    2016-01-01

    To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species. PMID:27304859

  7. Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation

    PubMed Central

    de Vries, Femke A.T.; de Boer, Esther; van den Bosch, Mike; Baarends, Willy M.; Ooms, Marja; Yuan, Li; Liu, Jian-Guo; van Zeeland, Albert A.; Heyting, Christa; Pastink, Albert

    2005-01-01

    In meiotic prophase, synaptonemal complexes (SCs) closely appose homologous chromosomes (homologs) along their length. SCs are assembled from two axial elements (AEs), one along each homolog, which are connected by numerous transverse filaments (TFs). We disrupted the mouse gene encoding TF protein Sycp1 to analyze the role of TFs in meiotic chromosome behavior and recombination. Sycp1-/- mice are infertile, but otherwise healthy. Sycp1-/- spermatocytes form normal AEs, which align homologously, but do not synapse. Most Sycp1-/- spermatocytes arrest in pachynema, whereas a small proportion reaches diplonema, or, exceptionally, metaphase I. In leptotene Sycp1-/- spermatocytes, γH2AX (indicative of DNA damage, including double-strand breaks) appears normal. In pachynema, Sycp1-/- spermatocytes display a number of discrete γH2AX domains along each chromosome, whereas γH2AX disappears from autosomes in wild-type spermatocytes. RAD51/DMC1, RPA, and MSH4 foci (which mark early and intermediate steps in pairing/recombination) appear in similar numbers as in wild type, but do not all disappear, and MLH1 and MLH3 foci (which mark late steps in crossing over) are not formed. Crossovers were rare in metaphase I of Sycp1-/- mice. We propose that SYCP1 has a coordinating role, and ensures formation of crossovers. Unexpectedly, Sycp1-/- spermatocytes did not form XY bodies. PMID:15937223

  8. Homologue engagement controls meiotic DNA break number and distribution.

    PubMed

    Thacker, Drew; Mohibullah, Neeman; Zhu, Xuan; Keeney, Scott

    2014-06-12

    Meiotic recombination promotes genetic diversification as well as pairing and segregation of homologous chromosomes, but the double-strand breaks (DSBs) that initiate recombination are dangerous lesions that can cause mutation or meiotic failure. How cells control DSBs to balance between beneficial and deleterious outcomes is not well understood. Here we test the hypothesis that DSB control involves a network of intersecting negative regulatory circuits. Using multiple complementary methods, we show that DSBs form in greater numbers in Saccharomyces cerevisiae cells lacking ZMM proteins, a suite of recombination-promoting factors traditionally regarded as acting strictly downstream of DSB formation. ZMM-dependent DSB control is genetically distinct from a pathway tying break formation to meiotic progression through the Ndt80 transcription factor. These counterintuitive findings suggest that homologous chromosomes that have successfully engaged one another stop making breaks. Genome-wide DSB maps uncover distinct responses by different subchromosomal domains to the ZMM mutation zip3 (also known as cst9), and show that Zip3 is required for the previously unexplained tendency of DSB density to vary with chromosome size. Thus, feedback tied to ZMM function contributes in unexpected ways to spatial patterning of recombination.

  9. Trans-regulation of mouse meiotic recombination hotspots by Rcr1.

    PubMed

    Parvanov, Emil D; Ng, Siemon H S; Petkov, Petko M; Paigen, Kenneth

    2009-02-17

    Meiotic recombination is required for the orderly segregation of chromosomes during meiosis and for providing genetic diversity among offspring. Among mammals, as well as yeast and higher plants, recombination preferentially occurs at highly delimited chromosomal sites 1-2 kb long known as hotspots. Although considerable progress has been made in understanding the roles various proteins play in carrying out the molecular events of the recombination process, relatively little is understood about the factors controlling the location and relative activity of mammalian recombination hotspots. To search for trans-acting factors controlling the positioning of recombination events, we compared the locations of crossovers arising in an 8-Mb segment of a 100-Mb region of mouse Chromosome 1 (Chr 1) when the longer region was heterozygous C57BL/6J (B6) x CAST/EiJ (CAST) and the remainder of the genome was either similarly heterozygous or entirely homozygous B6. The lack of CAST alleles in the remainder of the genome resulted in profound changes in hotspot activity in both females and males. Recombination activity was lost at several hotspots; new, previously undetected hotspots appeared; and still other hotspots remained unaffected, indicating the presence of distant trans-acting gene(s) whose CAST allele(s) activate or suppress the activity of specific hotspots. Testing the activity of three activated hotspots in sperm samples from individual male progeny of two genetic crosses, we identified a single trans-acting regulator of hotspot activity, designated Rcr1, that is located in a 5.30-Mb interval (11.74-17.04 Mb) on Chr 17. Using an Escherichia coli cloning assay to characterize the molecular products of recombination at two of these hotspots, we found that Rcr1 controls the appearance of both crossover and noncrossover gene conversion events, indicating that it likely controls the sites of the double-strand DNA breaks that initiate the recombination process.

  10. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  11. Dynamics of male meiotic recombination frequency during plant development using Fluorescent Tagged Lines in Arabidopsis thaliana.

    PubMed

    Li, Fan; De Storme, Nico; Geelen, Danny

    2017-02-13

    Meiotic homologous recombination plays a central role in creating genetic variability, making it an essential biological process relevant to evolution and crop breeding. In this study, we used pollen-specific fluorescent tagged lines (FTLs) to measure male meiotic recombination frequency during the development of Arabidopsis thaliana. Interestingly, a subset of pollen grains consistently shows loss of fluorescence expression in tested lines. Using nine independent FTL intervals, the spatio-temporal dynamics of male recombination frequency was assessed during plant development, considering both shoot type and plant age as independent parameters. In most genomic intervals assayed, male meiotic recombination frequency is highly consistent during plant development, showing no significant change between different shoot types and during plant aging. However, in some genomic regions, such as I1a and I5a, a small but significant effect of either developmental position or plant age were observed, indicating that the meiotic CO frequency in those intervals varies during plant development. Furthermore, from an overall view of all nine genomic intervals assayed, both primary and tertiary shoots show a similar dynamics of increasing recombination frequency during development, while secondary and lateral shoots remain highly stable. Our results provide new insights in the dynamics of male meiotic recombination frequency during plant development.

  12. Dynamics of male meiotic recombination frequency during plant development using Fluorescent Tagged Lines in Arabidopsis thaliana

    PubMed Central

    Li, Fan; De Storme, Nico; Geelen, Danny

    2017-01-01

    Meiotic homologous recombination plays a central role in creating genetic variability, making it an essential biological process relevant to evolution and crop breeding. In this study, we used pollen-specific fluorescent tagged lines (FTLs) to measure male meiotic recombination frequency during the development of Arabidopsis thaliana. Interestingly, a subset of pollen grains consistently shows loss of fluorescence expression in tested lines. Using nine independent FTL intervals, the spatio-temporal dynamics of male recombination frequency was assessed during plant development, considering both shoot type and plant age as independent parameters. In most genomic intervals assayed, male meiotic recombination frequency is highly consistent during plant development, showing no significant change between different shoot types and during plant aging. However, in some genomic regions, such as I1a and I5a, a small but significant effect of either developmental position or plant age were observed, indicating that the meiotic CO frequency in those intervals varies during plant development. Furthermore, from an overall view of all nine genomic intervals assayed, both primary and tertiary shoots show a similar dynamics of increasing recombination frequency during development, while secondary and lateral shoots remain highly stable. Our results provide new insights in the dynamics of male meiotic recombination frequency during plant development. PMID:28211906

  13. Immunofluorescent characterization of meiotic recombination in human males with variable spermatogenesis.

    PubMed

    de Vries, M; Ramos, L; de Boer, P

    2013-03-01

    Homologous recombination is the key to meiotic functioning. The basis of this process is provided by numerous SPO11-induced DNA double-strand breaks. Repair of these breaks occurs via the crossover (CO) and non-crossover (NCO) pathways. By means of immunofluorescence staining of Replication protein A (RPA) and MutL homolog 1 (MLH1) in combination with the DNA damage marker γH2AX, we studied transitional (CO and NCO) and late (CO) recombination nodules, respectively. Testicular samples were from non-obstructive azoospermic probands (testicular spermatozoa were found) and probands that had a history of normal fertility prior to a vasectomy. All probands were ICSI candidates. γH2AX foci mostly colocalized with delayed transitional nodules (RPA) for which variation was found among probands. Highest incidences of colocalization were found in patients. The level of MLH1 signal intensity was lower in probands who showed more frequent γH2AX RPA colocalization in late pachytene, suggesting communication between the CO and NCO pathways. Our results suggest the presence of a genetic risk pathway for children conceived from non-obstructive azoospermic probands and urge for follow-up studies investigating the level of recombination involved de novo mutations in these children.

  14. Evolutionary dynamics of meiotic recombination hotspots regulator PRDM9 in bovids.

    PubMed

    Ahlawat, Sonika; De, Sachinandan; Sharma, Priyanka; Sharma, Rekha; Arora, Reena; Kataria, R S; Datta, T K; Singh, R K

    2017-02-01

    Hybrid sterility or reproductive isolation in mammals has been attributed to allelic incompatibilities in a DNA-binding protein PRDM9. Not only is PRDM9 exceptional in being the only known 'speciation gene' in vertebrates, but it is also considered to be the fastest evolving gene in the genome. The terminal zinc finger (ZF) domain of PRDM9 specifies genome-wide meiotic recombination hotspot locations in mammals. Intriguingly, PRDM9 ZF domain is highly variable between as well as within species, possibly activating different recombination hotspots. The present study characterized the full-length coding sequence of PRDM9 in cattle and buffalo and explored the diversity of the ZF array in 514 samples from different bovids (cattle, yak, mithun, and buffalo). Substantial numerical and sequence variability were observed in the ZFs, with the number of repeats ranging from 6 to 9 in different bovines. Sequence analysis revealed the presence of 37 different ZFs in cattle, 3 in mithun, 4 in yak, and 13 in buffaloes producing 41 unique PRDM9 alleles in these species. The posterior mean of dN/dS or omega values calculated using Codeml tool of PAMLX identified sites -5, -1, +2, +3, +4, +5, and +6 in the ZF domain to be evolving positively in the studied species. Concerted evolution which typifies the evolution of this gene was consistently evident in all bovines. Our results demonstrate the extraordinary diversity of PRDM9 ZF array across bovines, reinforcing similar observations in other metazoans. The high variability is suggestive of unique repertoire of meiotic recombination hotspots in each species.

  15. The control of Spo11's interaction with meiotic recombination hotspots.

    PubMed

    Prieler, Silvia; Penkner, Alexandra; Borde, Valérie; Klein, Franz

    2005-01-15

    Programmed double-strand breaks (DSBs), which initiate meiotic recombination, arise through the activity of the evolutionary conserved topoisomerase homolog Spo11. Spo11 is believed to catalyze the DNA cleavage reaction in the initial step of DSB formation, while at least a further 11 factors assist in Saccharomyces cerevisiae. Using chromatin-immunoprecipitation (ChIP), we detected the transient, noncovalent association of Spo11 with meiotic hotspots in wild-type cells. The establishment of this association requires Rec102, Rec104, and Rec114, while the timely removal of Spo11 from chromatin depends on several factors, including Mei4 and Ndt80. In addition, at least one further component, namely, Red1, is responsible for locally restricting Spo11's interaction to the core region of the hotspot. In chromosome spreads, we observed meiosis-specific Spo11-Myc foci, independent of DSB formation, from leptotene until pachytene. In both rad50S and com1Delta/sae2Delta mutants, we observed a novel reaction intermediate between Spo11 and hotspots, which leads to the detection of full-length hotspot DNA by ChIP in the absence of artificial cross-linking. Although this DNA does not contain a break, its recovery requires Spo11's catalytic residue Y135. We propose that detection of uncross-linked full-length hotspot DNA is only possible during the reversible stage of the Spo11 cleavage reaction, in which rad50S and com1Delta/sae2Delta mutants transiently arrest.

  16. A Test of the Double-Strand Break Repair Model for Meiotic Recombination in Saccharomyces Cerevisiae

    PubMed Central

    Gilbertson, L. A.; Stahl, F. W.

    1996-01-01

    We tested predictions of the double-strand break repair (DSBR) model for meiotic recombination by examining the segregation patterns of small palindromic insertions, which frequently escape mismatch repair when in heteroduplex DNA. The palindromes flanked a well characterized DSB site at the ARG4 locus. The ``canonical'' DSBR model, in which only 5' ends are degraded and resolution of the four-stranded intermediate is by Holliday junction resolvase, predicts that hDNA will frequently occur on both participating chromatids in a single event. Tetrads reflecting this configuration of hDNA were rare. In addition, a class of tetrads not predicted by the canonical DSBR model was identified. This class represented events that produced hDNA in a ``trans'' configuration, on opposite strands of the same duplex on the two sides of the DSB site. Whereas most classes of convertant tetrads had typical frequencies of associated crossovers, tetrads with trans hDNA were parental for flanking markers. Modified versions of the DSBR model, including one that uses a topoisomerase to resolve the canonical DSBR intermediate, are supported by these data. PMID:8878671

  17. Association of poly-purine/poly-pyrimidine sequences with meiotic recombination hot spots

    PubMed Central

    Bagshaw, Andrew TM; Pitt, Joel PW; Gemmell, Neil J

    2006-01-01

    Background Meiotic recombination events have been found to concentrate in 1–2.5 kilo base regions, but these recombination hot spots do not share a consensus sequence and why they occur at specific sites is not fully understood. Some previous evidence suggests that poly-purine/poly-pyrimidine (poly-pu/py) tracts (PPTs), a class of sequence with distinctive biochemical properties, could be involved in recombination, but no general association of PPTs with meiotic recombination hot spots has previously been reported. Results We used computational methods to investigate in detail the relationship between PPTs and hot spots. We show statistical associations of PPT frequency with hot spots of meiotic recombination initiating lesions, double-strand breaks, in the genome of the yeast S. cerevisiae and with experimentally well characterized human meiotic recombination hot spots. Supporting a possible role of poly-pu/py-rich sequences in hot spot recombination, we also found that all three single nucleotide polymorphisms previously shown to be associated with human hot spot activity changes occur within sequence contexts of 14 bp or longer that are 85% or more poly-pu/py and at least 70% G/C. These polymorphisms are all close to the hot spot mid points. Comparing the sequences of experimentally characterized human hot spots with the orthologous regions of the chimpanzee genome previously shown not to contain hot spots, we found that in all five cases in which comparisons for the hot spot central regions are possible with publicly available sequence data, there are differences near the human hot spot mid points within sequences 14 bp or longer consisting of more than 80% poly-pu/py and at least 50% G/C. Conclusion Our results, along with previous evidence for the unique biochemical properties and recombination-stimulating potential of poly-pu/py-rich sequences, suggest that the possible functional involvement of this type of sequence in meiotic recombination hot spots

  18. PRDM9 drives evolutionary erosion of hotspots in Mus musculus through haplotype-specific initiation of meiotic recombination.

    PubMed

    Baker, Christopher L; Kajita, Shimpei; Walker, Michael; Saxl, Ruth L; Raghupathy, Narayanan; Choi, Kwangbom; Petkov, Petko M; Paigen, Kenneth

    2015-01-01

    Meiotic recombination generates new genetic variation and assures the proper segregation of chromosomes in gametes. PRDM9, a zinc finger protein with histone methyltransferase activity, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we measured activity of the Prdm9Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9Cst arose, and M.m. domesticus, into which Prdm9Cst was introduced experimentally. Comparing these two strains, we find that haplotype differences at hotspots lead to qualitative and quantitative changes in PRDM9 binding and activity. Using Mus spretus as an outlier, we found most variants affecting PRDM9Cst binding arose and were fixed in M.m. castaneus, suppressing hotspot activity. Furthermore, M.m. castaneus×M.m. domesticus F1 hybrids exhibit novel hotspots, with large haplotype biases in both PRDM9 binding and chromatin modification. These novel hotspots represent sites of historic evolutionary erosion that become activated in hybrids due to crosstalk between one parent's Prdm9 allele and the opposite parent's chromosome. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion.

  19. PRDM9 Drives Evolutionary Erosion of Hotspots in Mus musculus through Haplotype-Specific Initiation of Meiotic Recombination

    PubMed Central

    Baker, Christopher L.; Kajita, Shimpei; Walker, Michael; Saxl, Ruth L.; Raghupathy, Narayanan; Choi, Kwangbom; Petkov, Petko M.; Paigen, Kenneth

    2015-01-01

    Meiotic recombination generates new genetic variation and assures the proper segregation of chromosomes in gametes. PRDM9, a zinc finger protein with histone methyltransferase activity, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we measured activity of the Prdm9 Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9Cst arose, and M.m. domesticus, into which Prdm9Cst was introduced experimentally. Comparing these two strains, we find that haplotype differences at hotspots lead to qualitative and quantitative changes in PRDM9 binding and activity. Using Mus spretus as an outlier, we found most variants affecting PRDM9Cst binding arose and were fixed in M.m. castaneus, suppressing hotspot activity. Furthermore, M.m. castaneus×M.m. domesticus F1 hybrids exhibit novel hotspots, with large haplotype biases in both PRDM9 binding and chromatin modification. These novel hotspots represent sites of historic evolutionary erosion that become activated in hybrids due to crosstalk between one parent's Prdm9 allele and the opposite parent's chromosome. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion. PMID:25568937

  20. Abnormal meiotic recombination in infertile men and its association with sperm aneuploidy.

    PubMed

    Ferguson, Kyle A; Wong, Edgar Chan; Chow, Victor; Nigro, Mark; Ma, Sai

    2007-12-01

    Defects in early meiotic events are thought to play a critical role in male infertility; however, little is known regarding the relationship between early meiotic events and the chromosomal constitution of human sperm. Thus, we analyzed testicular tissue from 26 men (9 fertile and 17 infertile men), using immunofluorescent techniques to examine meiotic chromosomes, and fluorescent in situ hybridization to assess sperm aneuploidy. Based on a relatively small sample size, we observed that 42% (5/12) of men with impaired spermatogenesis displayed reduced genome-wide recombination when compared to the fertile men. Analysis of individual chromosomes showed chromosome-specific defects in recombination: chromosome 13 and 18 bivalents with only a single crossover and chromosome 21 bivalents lacking a crossover were more frequent among the infertile men. We identified two infertile men who displayed a novel meiotic defect in which the sex chromosomes failed to recombine: one man had an absence of sperm in the testes, while the other displayed increased sex chromosome aneuploidy in the sperm, resulting in a 45,X abortus after intracytoplasmic sperm injection. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. Recombination between the sex chromosomes may be a useful indicator for identifying men at risk of producing chromosomally abnormal sperm. An understanding of the molecular mechanisms that contribute to sperm aneuploidy in infertile men could aid in risk assessment for couples undergoing assisted reproduction.

  1. Chromosome Synapsis Alleviates Mek1-Dependent Suppression of Meiotic DNA Repair

    PubMed Central

    Subramanian, Vijayalakshmi V.; MacQueen, Amy J.; Vader, Gerben; Shinohara, Miki; Sanchez, Aurore; Borde, Valérie; Shinohara, Akira; Hochwagen, Andreas

    2016-01-01

    Faithful meiotic chromosome segregation and fertility require meiotic recombination between homologous chromosomes rather than the equally available sister chromatid, a bias that in Saccharomyces cerevisiae depends on the meiotic kinase, Mek1. Mek1 is thought to mediate repair template bias by specifically suppressing sister-directed repair. Instead, we found that when Mek1 persists on closely paired (synapsed) homologues, DNA repair is severely delayed, suggesting that Mek1 suppresses any proximal repair template. Accordingly, Mek1 is excluded from synapsed homologues in wild-type cells. Exclusion requires the AAA+-ATPase Pch2 and is directly coupled to synaptonemal complex assembly. Stage-specific depletion experiments further demonstrate that DNA repair in the context of synapsed homologues requires Rad54, a repair factor inhibited by Mek1. These data indicate that the sister template is distinguished from the homologue primarily by its closer proximity to inhibitory Mek1 activity. We propose that once pairing or synapsis juxtaposes homologues, exclusion of Mek1 is necessary to avoid suppression of all templates and accelerate repair progression. PMID:26870961

  2. Together yes, but not coupled: new insights into the roles of RAD51 and DMC1 in plant meiotic recombination.

    PubMed

    Pradillo, Mónica; López, Eva; Linacero, Rosario; Romero, Concepción; Cuñado, Nieves; Sánchez-Morán, Eugenio; Santos, Juan L

    2012-03-01

    The eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, non-homologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.

  3. The Rec102 Mutant of Yeast Is Defective in Meiotic Recombination and Chromosome Synapsis

    PubMed Central

    Bhargava, J.; Engebrecht, J. A.; Roeder, G. S.

    1992-01-01

    A mutation at the REC102 locus was identified in a screen for yeast mutants that produce inviable spores. rec102 spore lethality is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The rec102 mutation completely eliminates meiotically induced gene conversion and crossing over but has no effect on mitotic recombination frequencies. Cytological studies indicate that the rec102 mutant makes axial elements (precursors to the synaptonemal complex), but homologous chromosomes fail to synapse. In addition, meiotic chromosome segregation is significantly delayed in rec102 strains. Studies of double and triple mutants indicate that the REC102 protein acts before the RAD52 gene product in the meiotic recombination pathway. The REC102 gene was cloned based on complementation of the mutant defect and the gene was mapped to chromosome XII between CDC25 and STE11. PMID:1732169

  4. Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast.

    PubMed

    Lam, Isabel; Mohibullah, Neeman; Keeney, Scott

    2017-01-01

    Meiosis is a specialized form of cell division resulting in reproductive cells with a reduced, usually haploid, genome complement. A key step after premeiotic DNA replication is the occurrence of homologous recombination at multiple places throughout the genome, initiated with the formation of DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like protein Spo11. DSBs are distributed non-randomly in genomes, and understanding the mechanisms that shape this distribution is important for understanding how meiotic recombination influences heredity and genome evolution. Several methods exist for mapping where Spo11 acts. Of these, sequencing of Spo11-associated oligonucleotides (Spo11 oligos) is the most precise, specifying the locations of DNA breaks to the base pair. In this chapter we detail the steps involved in Spo11-oligo mapping in the SK1 strain of budding yeast Saccharomyces cerevisiae, from harvesting cells of highly synchronous meiotic cultures, through preparation of sequencing libraries, to the mapping pipeline used for processing the data.

  5. Gradual implementation of the meiotic recombination program via checkpoint pathways controlled by global DSB levels.

    PubMed

    Joshi, Neeraj; Brown, M Scott; Bishop, Douglas K; Börner, G Valentin

    2015-03-05

    During meiosis, Spo11-induced double-strand breaks (DSBs) are processed into crossovers, ensuring segregation of homologous chromosomes (homologs). Meiotic DSB processing entails 5' end resection and preferred strand exchange with the homolog rather than the sister chromatid (homolog bias). In many organisms, DSBs appear gradually along the genome. Here we report unexpected effects of global DSB levels on local recombination events. Early-occurring, low-abundance "scout" DSBs lack homolog bias. Their resection and interhomolog processing are controlled by the conserved checkpoint proteins Tel1(ATM) kinase and Pch2(TRIP13) ATPase. Processing pathways controlled by Mec1(ATR) kinase take over these functions only above a distinct DSB threshold, resulting in progressive strengthening of the homolog bias. We conclude that Tel1(ATM)/Pch2 and Mec1(ATR) DNA damage response pathways are sequentially activated during wild-type meiosis because of their distinct sensitivities to global DSB levels. Moreover, relative DSB order controls the DSB repair pathway choice and, ultimately, recombination outcome.

  6. Polyploidization increases meiotic recombination frequency in Arabidopsis: a close look at statistical modeling and data analysis.

    PubMed

    Wang, Lin; Luo, Zewei

    2012-04-18

    This paper is a response to Pecinka A, Fang W, Rehmsmeier M, Levy AA, Mittelsten Scheid, O: Polyploidization increases meiotic recombination frequency in Arabidopsis. BMC Biology 2011, 9:24.See research article at http://www.biomedcentral.com/1741-7007/9/24.

  7. Meiotic recombination errors, the origin of sperm aneuploidy and clinical recommendations.

    PubMed

    Tempest, Helen G

    2011-02-01

    Since the early 1990s male infertility has successfully been treated by intracytoplasmic sperm injection (ICSI), nevertheless concerns have been raised regarding the genetic risk of ICSI. Chromosome aneuploidy (the presence of extra or missing chromosomes) is the leading cause of pregnancy loss and mental retardation in humans. While the majority of chromosome aneuploidies are maternal in origin, the paternal contribution to aneuploidy is clinically relevant particularly for the sex chromosomes. Given that it is difficult to study female gametes investigations are predominantly conducted in male meiotic recombination and sperm aneuploidy. Research suggests that infertile men have increased levels of sperm aneuploidy and that this is likely due to increased errors in meiotic recombination and chromosome synapsis within these individuals. It is perhaps counterintuitive but there appears to be no selection against chromosomally aneuploid sperm at fertilization. In fact the frequency of aneuploidy in sperm appears to be mirrored in conceptions. Given this information this review will cover our current understanding of errors in meiotic recombination and chromosome synapsis and how these may contribute to increased sperm aneuploidy. Frequencies of sperm aneuploidy in infertile men and individuals with constitutional karyotypic abnormalities are reviewed, and based on these findings, indications for clinical testing of sperm aneuploidy are discussed. In addition, the application of single nucleotide arrays for the analysis of meiotic recombination and identification of parental origin of aneuploidy are considered.

  8. Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR).

    PubMed

    Gray, Stephen; Allison, Rachal M; Garcia, Valerie; Goldman, Alastair S H; Neale, Matthew J

    2013-07-31

    During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation.

  9. Targeted induction of meiotic double-strand breaks reveals chromosomal domain-dependent regulation of Spo11 and interactions among potential sites of meiotic recombination.

    PubMed

    Fukuda, Tomoyuki; Kugou, Kazuto; Sasanuma, Hiroyuki; Shibata, Takehiko; Ohta, Kunihiro

    2008-02-01

    Meiotic recombination is initiated by programmed DNA double-strand break (DSB) formation mediated by Spo11. DSBs occur with frequency in chromosomal regions called hot domains but are seldom seen in cold domains. To obtain insights into the determinants of the distribution of meiotic DSBs, we examined the effects of inducing targeted DSBs during yeast meiosis using a UAS-directed form of Spo11 (Gal4BD-Spo11) and a meiosis-specific endonuclease, VDE (PI-SceI). Gal4BD-Spo11 cleaved its target sequence (UAS) integrated in hot domains but rarely in cold domains. However, Gal4BD-Spo11 did bind to UAS and VDE efficiently cleaved its recognition sequence in either context, suggesting that a cold domain is not a region of inaccessible or uncleavable chromosome structure. Importantly, self-association of Spo11 occurred at UAS in a hot domain but not in a cold domain, raising the possibility that Spo11 remains in an inactive intermediate state in cold domains. Integration of UAS adjacent to known DSB hotspots allowed us to detect competitive interactions among hotspots for activation. Moreover, the presence of VDE-introduced DSB repressed proximal hotspot activity, implicating DSBs themselves in interactions among hotspots. Thus, potential sites for Spo11-mediated DSB are subject to domain-specific and local competitive regulations during and after DSB formation.

  10. Human meiotic recombination products revealed by sequencing a hotspot for homologous strand exchange in multiple HNPP deletion patients.

    PubMed

    Reiter, L T; Hastings, P J; Nelis, E; De Jonghe, P; Van Broeckhoven, C; Lupski, J R

    1998-05-01

    The HNPP (hereditary neuropathy with liability to pressure palsies) deletion and CMT1A (Charcot-Marie-Tooth disease type 1A) duplication are the reciprocal products of homologous recombination events between misaligned flanking CMT1A-REP repeats on chromosome 17p11. 2-p12. A 1.7-kb hotspot for homologous recombination was previously identified wherein the relative risk of an exchange event is 50 times higher than in the surrounding 98.7% identical sequence shared by the CMT1A-REPs. To refine the region of exchange further, we designed a PCR strategy to amplify the recombinant CMT1A-REP from HNPP patients as well as the proximal and distal CMT1A-REPs from control individuals. By comparing the sequences across recombinant CMT1A-REPs to that of the proximal and distal CMT1A-REPs, the exchange was mapped to a 557-bp region within the previously identified 1.7-kb hotspot in 21 of 23 unrelated HNPP deletion patients. Two patients had recombined sequences suggesting an exchange event closer to the mariner-like element previously identified near the hotspot. Five individuals also had interspersed patches of proximal or distal repeat specific DNA sequence indicating potential gene conversion during the exchange of genetic material. Our studies provide a direct observation of human meiotic recombination products. These results are consistent with the hypothesis that minimum efficient processing segments, which have been characterized in Escherichia coli, yeast, and cultured mammalian cells, may be required for efficient homologous meiotic recombination in humans.

  11. The Utilization during Mitotic Cell Division of Loci Controlling Meiotic Recombination and Disjunction in DROSOPHILA MELANOGASTER

    PubMed Central

    Baker, Bruce S.; Carpenter, Adelaide T. C.; Ripoll, P.

    1978-01-01

    To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by UV and X rays.—Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells.—The chromosome instability produced by mei-41 alleles is the consequence of chromosome breakage, that of mei-9 alleles is primarily due to chromosome breakage and, to a lesser extent, to an elevated frequency of mitotic recombination, whereas no predominant mechanism responsible for the instability caused by c(3)G alleles is discernible. Since these three loci are defective in their responses to mutagen damage, their effects on chromosome stability in nonmutagenized cells are interpreted as resulting from an inability to repair spontaneous lesions. Both mei-W68 and mei-S282 increase mitotic recombination (and in mei-W68, to a lesser extent, chromosome loss) in the abdomen but not the wing. In the abdomen, the primary effect on chromosome stability occurs during the larval period when the abdominal histoblasts

  12. Fine-scale variation in meiotic recombination in Mimulus inferred from population shotgun sequencing

    SciTech Connect

    Hellsten, Uffe; Wright, Kevin M.; Jenkins, Jerry; Shu, Shengqiang; Yuan, Yao-Wu; Wessler, Susan R.; Schmutz, Jeremy; Willis, John H.; Rokhsar, Daniel S.

    2013-11-13

    Meiotic recombination rates can vary widely across genomes, with hotspots of intense activity interspersed among cold regions. In yeast, hotspots tend to occur in promoter regions of genes, whereas in humans and mice hotspots are largely defined by binding sites of the PRDM9 protein. To investigate the detailed recombination pattern in a flowering plant we use shotgun resequencing of a wild population of the monkeyflower Mimulus guttatus to precisely locate over 400,000 boundaries of historic crossovers or gene conversion tracts. Their distribution defines some 13,000 hotspots of varying strengths, interspersed with cold regions of undetectably low recombination. Average recombination rates peak near starts of genes and fall off sharply, exhibiting polarity. Within genes, recombination tracts are more likely to terminate in exons than in introns. The general pattern is similar to that observed in yeast, as well as in PRDM9-knockout mice, suggesting that recombination initiation described here in Mimulus may reflect ancient and conserved eukaryotic mechanisms

  13. The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis.

    PubMed

    Knoll, Alexander; Higgins, James D; Seeliger, Katharina; Reha, Sarah J; Dangel, Natalie J; Bauknecht, Markus; Schröpfer, Susan; Franklin, F Christopher H; Puchta, Holger

    2012-04-01

    The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.

  14. Meiotic recombination in normal and cloned bulls and their offspring

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Homologous chromosome pairing and recombination are essential components of meiosis and sexual reproduction. The reshuffling of genetic material through breakage and reunion of chromatids ensure proper segregation of homologous chromosomes in reduction division and genetic diversity in the progeny....

  15. Segregation of yeast polymorphic STA genes in meiotic recombinants and analysis of glucoamylase production.

    PubMed

    Balogh, I; Maráz, A

    1996-12-01

    Hybrid yeast strains were constructed using haploid Saccharomyces cerevisiae and Saccharomyces cerevisiae var. diastaticus strains to get haploid meiotic recombinants having more than one copy of STA1, STA2, and STA3 genes. STA genes were localized on the chromosomes by pulsed field gel electrophoresis. Working gene dosage effects were found among STA genes in liquid starch medium, indicating low levels of glucose repression. Growth of strains, however, was not influenced by their STA copy number.

  16. The synaptonemal complex and meiotic recombination in humans: new approaches to old questions.

    PubMed

    Vallente, Rhea U; Cheng, Edith Y; Hassold, Terry J

    2006-06-01

    Meiotic prophase serves as an arena for the interplay of two important cellular activities, meiotic recombination and synapsis of homologous chromosomes. Synapsis is mediated by the synaptonemal complex (SC), originally characterized as a structure linked to pairing of meiotic chromosomes (Moses (1958) J Biophys Biochem Cytol 4:633-638). In 1975, the first electron micrographs of human pachytene stage SCs were presented (Moses et al. (1975) Science 187:363-365) and over the next 15 years the importance of the SC to normal meiotic progression in human males and females was established (Jhanwar and Chaganti (1980) Hum Genet 54:405-408; Pathak and Elder (1980) Hum Genet 54:171-175; Solari (1980) Chromosoma 81:315-337; Speed (1984) Hum Genet 66:176-180; Wallace and Hulten (1985) Ann Hum Genet 49(Pt 3):215-226). Further, these studies made it clear that abnormalities in the assembly or maintenance of the SC were an important contributor to human infertility (Chaganti et al. (1980) Am J Hum Genet 32:833-848; Vidal et al. (1982) Hum Genet 60:301-304; Bojko (1983) Carlsberg Res Commun 48:285-305; Bojko (1985) Carlsberg Res Commun 50:43-72; Templado et al. (1984) Hum Genet 67:162-165; Navarro et al. (1986) Hum Reprod 1:523-527; Garcia et al. (1989) Hum Genet 2:147-53). However, the utility of these early studies was limited by lack of information on the structural composition of the SC and the identity of other SC-associated proteins. Fortunately, studies of the past 15 years have gone a long way toward remedying this problem. In this minireview, we highlight the most important of these advances as they pertain to human meiosis, focusing on temporal aspects of SC assembly, the relationship between the SC and meiotic recombination, and the contribution of SC abnormalities to human infertility.

  17. H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation

    PubMed Central

    Xu, Zhiliang; Song, Zhenhua; Li, Guoping; Tu, Huayu; Liu, Weixiao; Liu, Yujiao; Wang, Pan; Wang, Yuanting; Cui, Xiuhong; Liu, Chao; Shang, Yongliang; de Rooij, Dirk G.; Gao, Fei; Li, Wei

    2016-01-01

    Meiotic recombination is essential for fertility in most sexually reproducing species, but the molecular mechanisms underlying this process remain poorly understood in mammals. Here, we show that RNF20-mediated H2B ubiquitination is required for meiotic recombination. A germ cell-specific knockout of the H2B ubiquitination E3 ligase RNF20 results in complete male infertility. The Stra8-Rnf20−/− spermatocytes arrest at the pachytene stage because of impaired programmed double-strand break (DSB) repair. Further investigations reveal that the depletion of RNF20 in the germ cells affects chromatin relaxation, thus preventing programmed DSB repair factors from being recruited to proper positions on the chromatin. The gametogenetic defects of the H2B ubiquitination deficient cells could be partially rescued by forced chromatin relaxation. Taken together, our results demonstrate that RNF20/Bre1p-mediated H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation, and suggest an old drug may provide a new way to treat some oligo- or azoospermia patients with chromatin relaxation disorders. PMID:27431324

  18. Meiotic prophase roles of Rec8 in crossover recombination and chromosome structure

    PubMed Central

    Yoon, Sang-Wook; Lee, Min-Su; Xaver, Martin; Zhang, Liangran; Hong, Soo-Gil; Kong, Yoon-Ju; Cho, Hong-Rae; Kleckner, Nancy; Kim, Keun P.

    2016-01-01

    Rec8 is a prominent component of the meiotic prophase chromosome axis that mediates sister chromatid cohesion, homologous recombination and chromosome synapsis. Here, we explore the prophase roles of Rec8. (i) During the meiotic divisions, Rec8 phosphorylation mediates its separase-mediated cleavage. We show here that such cleavage plays no detectable role for chromosomal events of prophase. (ii) We have analyzed in detail three rec8 phospho-mutants, with 6, 24 or 29 alanine substitutions. A distinct ‘separation of function’ phenotype is revealed. In the mutants, axis formation and recombination initiation are normal, as is non-crossover recombination; in contrast, crossover (CO)-related events are defective. Moreover, the severities of these defects increase coordinately with the number of substitution mutations, consistent with the possibility that global phosphorylation of Rec8 is important for these effects. (iii) We have analyzed the roles of three kinases that phosphorylate Rec8 during prophase. Timed inhibition of Dbf4-dependent Cdc7 kinase confers defects concordant with rec8 phospho-mutant phenotypes. Inhibition of Hrr25 or Cdc5/polo-like kinase does not. Our results suggest that Rec8's prophase function, independently of cohesin cleavage, contributes to CO-specific events in conjunction with the maintenance of homolog bias at the leptotene/zygotene transition of meiotic prophase. PMID:27484478

  19. The mouse Eb meiotic recombination hotspot contains a tissue-specific transcriptional enhancer.

    PubMed

    Ling, X; Shenkar, R; Sakai, D; Arnheim, N

    1993-01-01

    A meiotic recombination hotspot exists within the second intron of the mouse major histocompatibility complex (MHC) gene, Eb. In the present study, a small fragment from the intron which contains two potential transcriptional regulatory elements was cloned into an expression vector and its effect on transcription was tested. This fragment was found to contain tissue-specific transcriptional enhancer activity. An octamer-like sequence and a B motif may contribute to this enhancer activity. Similar regulatory sequences with the same orientation and distance from one another are found in another mouse MHC recombination hotspot.

  20. Increased frequency of asynapsis and associated meiotic silencing of heterologous chromatin in the presence of irradiation-induced extra DNA double strand breaks.

    PubMed

    Schoenmakers, Sam; Wassenaar, Evelyne; van Cappellen, Wiggert A; Derijck, Alwin A; de Boer, Peter; Laven, Joop S E; Grootegoed, J Anton; Baarends, Willy M

    2008-05-01

    In meiotic prophase of male placental mammals, the heterologous X and Y chromosomes remain largely unsynapsed, which activates meiotic sex chromosome inactivation (MSCI), leading to formation of the transcriptionally silenced XY body. MSCI is most likely related to meiotic silencing of unsynapsed chromatin (MSUC), a mechanism that can silence autosomal unsynapsed chromatin. However, heterologous synapsis and escape from silencing also occur. In mammalian species, formation of DNA double strand breaks (DSBs) during leptotene precedes meiotic chromosome pairing. These DSBs are essential to achieve full synapsis of homologous chromosomes. We generated 25% extra meiotic DSBs by whole body irradiation of mice. This leads to a significant increase in meiotic recombination frequency. In mice carrying translocation chromosomes with synaptic problems, we observed an approximately 35% increase in asynapsis and MSUC of the nonhomologous region in the smallest chromosome pair following irradiation. However, the same nonhomologous region in the largest chromosome pair, shows complete synapsis and escape from MSUC in almost 100% of the nuclei, irrespective of exposure to irradiation. We propose that prevention of synapsis and associated activation of MSUC is linked to the presence of unrepaired meiotic DSBs in the nonhomologous region. Also, spreading of synaptonemal complex formation from regions of homology may act as an opposing force, and drive heterologous synapsis.

  1. Histone H3 lysine 4 trimethylation marks meiotic recombination initiation sites.

    PubMed

    Borde, Valérie; Robine, Nicolas; Lin, Waka; Bonfils, Sandrine; Géli, Vincent; Nicolas, Alain

    2009-01-21

    The function of histone modifications in initiating and regulating the chromosomal events of the meiotic prophase remains poorly understood. In Saccharomyces cerevisiae, we examined the genome-wide localization of histone H3 lysine 4 trimethylation (H3K4me3) along meiosis and its relationship to gene expression and position of the programmed double-strand breaks (DSBs) that initiate interhomologue recombination, essential to yield viable haploid gametes. We find that the level of H3K4me3 is constitutively higher close to DSB sites, independently of local gene expression levels. Without Set1, the H3K4 methylase, 84% of the DSB sites exhibit a severely reduced DSB frequency, the reduction being quantitatively correlated with the local level of H3K4me3 in wild-type cells. Further, we show that this differential histone mark is already established in vegetative cells, being higher in DSB-prone regions than in regions with no or little DSB. Taken together, our results demonstrate that H3K4me3 is a prominent and preexisting mark of active meiotic recombination initiation sites. Novel perspectives to dissect the various layers of the controls of meiotic DSB formation are discussed.

  2. MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice[OPEN

    PubMed Central

    Wang, Chong; Yu, Junping; Zong, Jie; Lu, Pingli

    2016-01-01

    F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression. PMID:27436711

  3. Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11.

    PubMed

    Nichols, M D; DeAngelis, K; Keck, J L; Berger, J M

    1999-11-01

    In all organisms, type II DNA topoisomerases are essential for untangling chromosomal DNA. We have determined the structure of the DNA-binding core of the Methanococcus jannaschii DNA topoisomerase VI A subunit at 2.0 A resolution. The overall structure of this subunit is unique, demonstrating that archaeal type II enzymes are distinct from other type II topoisomerases. However, the core structure contains a pair of domains that are also found in type IA and classic type II topoisomerases. Together, these regions may form the basis of a DNA cleavage mechanism shared among these enzymes. The core A subunit is a dimer that contains a deep groove that spans both protomers. The dimer architecture suggests that DNA is bound in the groove, across the A subunit interface, and that the two monomers separate during DNA transport. The A subunit of topoisomerase VI is homologous to the meiotic recombination factor, Spo11, and this structure can serve as a template for probing Spo11 function in eukaryotes.

  4. DNA double-strand breaks, but not crossovers, are required for the reorganization of meiotic nuclei in Tetrahymena

    PubMed Central

    Mochizuki, Kazufumi; Novatchkova, Maria; Loidl, Josef

    2011-01-01

    Summary During meiosis, the micronuclei of the ciliated protist Tetrahymena thermophila elongate dramatically. Within these elongated nuclei, chromosomes are arranged in a bouquet-like fashion and homologous pairing and recombination takes place. We studied meiotic chromosome behavior in Tetrahymena in the absence of two genes, SPO11 and a homolog of HOP2 (HOP2A), which have conserved roles in the formation of meiotic DNA double-strand breaks (DSBs) and their repair, respectively. Single-knockout mutants for each gene display only a moderate reduction in chromosome pairing, but show a complete failure to form chiasmata and exhibit chromosome missegregation. The lack of SPO11 prevents the elongation of meiotic nuclei, but it is restored by the artificial induction of DSBs. In the hop2AΔ mutant, the transient appearance of γ-H2A.X and Rad51p signals indicates the formation and efficient repair of DSBs; but this repair does not occur by interhomolog crossing over. In the absence of HOP2A, the nuclei are elongated, meaning that DSBs but not their conversion to crossovers are required for the development of this meiosis-specific morphology. In addition, by in silico homology searches, we compiled a list of likely Tetrahymena meiotic proteins as the basis for further studies of the unusual synaptonemal complex-less meiosis in this phylogenetically remote model organism. PMID:18522989

  5. Abnormal meiotic recombination with complex chromosomal rearrangement in an azoospermic man.

    PubMed

    Wang, Liu; Iqbal, Furhan; Li, Guangyuan; Jiang, Xiaohua; Bukhari, Ihtisham; Jiang, Hanwei; Yang, Qingling; Zhong, Liangwen; Zhang, Yuanwei; Hua, Juan; Cooke, Howard J; Shi, Qinghua

    2015-06-01

    Spermatocyte spreading and immunostaining were applied to detect meiotic prophase I progression, homologous chromosome pairing, synapsis and recombination in an azoospermic reciprocal translocation 46, XY, t(5;7;9;13)(5q11;7p11;7p15;9q12;13p12) carrier. Histological examination of the haematoxylin and eosin stained testicular sections revealed reduced germ cells with no spermatids or sperm in the patient. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay showed apoptotic cells in testicular sections of translocation carrier. Immnunofluorescence analysis indicated the presence of an octavalent in all the pachytene spermatocytes analysed in the patient. Meiotic progression was disturbed, as an increase in zygotene (P < 0.001) and decrease in the pachytene spermatocytes (P < 0.001) were observed in the t(5;7;9;13) carrier compared with controls. It was further observed that 93% of octavalents were found partially asynapsed between homologous chromosomes. A significant decrease in the recombination frequency was observed on 5p, 5q, 7q, 9p and 13q in the translocation carrier compared with the reported controls. A significant reduction in XY recombination frequency was also found in the participants. Our results indicated that complex chromosomal rearrangements can impair synaptic integrity of translocated chromosomes, which may reduce chromosomal recombination on translocated as well as non-translocated chromosomes, a phenomenon commonly known as interchromosomal effect.

  6. Three Decades of Recombinant DNA.

    ERIC Educational Resources Information Center

    Palmer, Jackie

    1985-01-01

    Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

  7. Evidence for human meiotic recombination interference obtained through construction of a short tandem repeat-polymorphism linkage map of chromosome 19

    SciTech Connect

    Weber, J.L.; Wang, Z.; Hansen, K.; Stephenson, M.; Kappel, C.; Salzman, S.; Wilkie, P.J. ); Keats, B. ); Dracopoli, N.C. ); Brandriff, B.F.; Olsen, A.S. )

    1993-11-01

    An improved linkage map for human chromosome 19 containing 35 short tandem repeat polymorphisms (STRPs) and one VNTR (D19S20) was constructed. The map included 12 new (GATA)[sub n] tetranucleotide STRPs. Although total lengths of the male (114 cM) and female (128 cM) maps were similar, at both ends of the chromosome male recombination exceeded female recombination, while in the interior portion of the map female recombination was in excess. Cosmid clones containing the STRP sequences were identified and were positioned along the chromosome by fluorescent in situ hybridization. Four rounds of careful checking and removal of genotyping errors allowed biologically relevant conclusions to be made concerning the numbers and distributions of recombination events on chromosome 19. The average numbers of recombinations per chromosome matched closely the lengths of the genetic maps computed by using the program CRIMAP. Significant numbers of chromosomes with zero, one, two, or three recombinations were detected as products of both female and male meioses. On the basis of the total number of observed pairs of recombination events in which only a single informative marker was situated between the two recombinations, a maximal estimate for the rate of meiotic STRP [open quotes]gene[close quotes] conversion without recombination was calculated as 3 [times] 10[sup [minus]4]/meiosis. For distances up to 30 cM between recombinations, many fewer chromosomes which had undergone exactly two recombinations were observed than were expected on the basis of the assumption of independent recombination locations. This strong new evidence for human meiotic interference will help to improve the accuracy of interpretation of clinical DNA test results involving polymorphisms flanking a genetic abnormality. 61 refs., 2 figs., 5 tabs.

  8. Recombination spots prediction using DNA physical properties in the saccharomyces cerevisiae genome

    NASA Astrophysics Data System (ADS)

    Guo, Shou-Hui; Xu, Li-Qin; Chen, Wei; Liu, Guo-Qing; Lin, Hao

    2012-09-01

    The prediction of meiotic recombination is difficult and current available methods are limited. In this study, we propose a novel method for discriminating between recombination hotspots and coldspots using support vector machine(SVM) with the DNA physical properties. Results of optimized pseudo-tetranucleotide show overall accuracy of 83.1% by using 5-fold cross-validation. High predictive successful rate exhibit that this model can be applied for discriminating between recombination hotspots and coldspots.

  9. Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis

    PubMed Central

    Francis, Kirk E.; Lam, Sandy Y.; Harrison, Benjamin D.; Bey, Alexandra L.; Berchowitz, Luke E.; Copenhaver, Gregory P.

    2007-01-01

    Recombination, in the form of cross-overs (COs) and gene conversion (GC), is a highly conserved feature of meiosis from fungi to mammals. Recombination helps ensure chromosome segregation and promotes allelic diversity. Lesions in the recombination machinery are often catastrophic for meiosis, resulting in sterility. We have developed a visual assay capable of detecting Cos and GCs and measuring CO interference in Arabidopsis thaliana. This flexible assay utilizes transgene constructs encoding pollen-expressed fluorescent proteins of three different colors in the qrt1 mutant background. By observing the segregation of the fluorescent alleles in 92,489 pollen tetrads, we demonstrate (i) a correlation between developmental position and CO frequency, (ii) a temperature dependence for CO frequency, (iii) the ability to detect meiotic GC events, and (iv) the ability to rapidly assess CO interference. PMID:17360452

  10. Probing Meiotic Recombination and Aneuploidy of Single Sperm Cells by Whole Genome Sequencing

    PubMed Central

    Lu, Sijia; Zong, Chenghang; Fan, Wei; Yang, Mingyu; Li, Jinsen; Chapman, Alec R.; Zhu, Ping; Hu, Xuesong; Xu, Liya; Yan, Liying; Bai, Fan; Qiao, Jie; Tang, Fuchou; Li, Ruiqiang; Xie, X. Sunney

    2013-01-01

    Meiotic recombination creates genetic diversity and ensures segregation of homologous chromosomes. Previous population analyses yielded results averaged among individuals and impacted by evolutionary pressures. Here we sequenced 99 sperm from an Asian male using the newly developed amplification method—Multiple Annealing and Looping-Based Amplification Cycles (MALBAC)—to phase the personal genome and map at high resolution recombination events, which are non-uniformly distributed across the genome in the absence of selection pressure. The paucity of recombination near transcription start sites observed in individual sperm indicates such a phenomenon is intrinsic to the molecular mechanism of meiosis. Interestingly, a decreased crossover frequency in companion with an increase of autosomal aneuploidy is observable on a global per-sperm basis. PMID:23258895

  11. Antagonistic roles of ubiquitin ligase HEI10 and SUMO ligase RNF212 regulate meiotic recombination.

    PubMed

    Qiao, Huanyu; Prasada Rao, H B D; Yang, Ye; Fong, Jared H; Cloutier, Jeffrey M; Deacon, Dekker C; Nagel, Kathryn E; Swartz, Rebecca K; Strong, Edward; Holloway, J Kim; Cohen, Paula E; Schimenti, John; Ward, Jeremy; Hunter, Neil

    2014-02-01

    Crossover recombination facilitates the accurate segregation of homologous chromosomes during meiosis. In mammals, poorly characterized regulatory processes ensure that every pair of chromosomes obtains at least one crossover, even though most recombination sites yield non-crossovers. Designation of crossovers involves selective localization of the SUMO ligase RNF212 to a minority of recombination sites, where it stabilizes pertinent factors such as MutSγ (ref. 4). Here we show that the ubiquitin ligase HEI10 (also called CCNB1IP1) is essential for this crossover/non-crossover differentiation process. In HEI10-deficient mice, RNF212 localizes to most recombination sites, and dissociation of both RNF212 and MutSγ from chromosomes is blocked. Consequently, recombination is impeded, and crossing over fails. In wild-type mice, HEI10 accumulates at designated crossover sites, suggesting that it also has a late role in implementing crossing over. As with RNF212, dosage sensitivity for HEI10 indicates that it is a limiting factor for crossing over. We suggest that SUMO and ubiquitin have antagonistic roles during meiotic recombination that are balanced to effect differential stabilization of recombination factors at crossover and non-crossover sites.

  12. Sites of strong Rec12/Spo11 binding in the fission yeast genome are associated with meiotic recombination and with centromeres

    PubMed Central

    Ludin, Katja; Mata, Juan; Watt, Stephen; Lehmann, Elisabeth; Bähler, Jürg; Kohli, Jürg

    2013-01-01

    Meiotic recombination arises from Rec12/Spo11-dependent formation of DNA double-strand breaks (DSBs) and their subsequent repair. We identified Rec12-binding peaks across the S. pombe genome using chromatin immunoprecipitation after reversible formaldehyde cross-linking combined with whole-genome DNA microarrays. Strong Rec12-binding coincided with previously identified DSBs at the recombination hotspots ura4A, mbs1, and mbs2, and correlated with DSB formation at a new site. In addition, Rec12-binding corresponded to eight novel conversion hotspots and correlated with crossover density in segments of chromosome I. Notably, Rec12-binding inversely correlated with GC content, contrary to findings in S. cerevisiae. Although both replication origins and Rec12-binding sites preferred AT-rich gene-free regions, they seemed to exclude each other. We also uncovered a connection between binding sites of Rec12 and meiotic cohesin Rec8. Rec12-binding peaks lay often within 2.5 kb of a Rec8-binding peak. Rec12-binding showed preference for large intergenic regions and was found to bind preferentially near to genes expressed strongly in meiosis. Surprisingly, Rec12-binding was also detected in centromeric core regions, which raises the intriguing possibility that Rec12 plays additional roles in meiotic chromosome dynamics. PMID:18449558

  13. Sites of strong Rec12/Spo11 binding in the fission yeast genome are associated with meiotic recombination and with centromeres.

    PubMed

    Ludin, Katja; Mata, Juan; Watt, Stephen; Lehmann, Elisabeth; Bähler, Jürg; Kohli, Jürg

    2008-10-01

    Meiotic recombination arises from Rec12/Spo11-dependent formation of DNA double-strand breaks (DSBs) and their subsequent repair. We identified Rec12-binding peaks across the Schizosaccharomyces pombe genome using chromatin immunoprecipitation after reversible formaldehyde cross-linking combined with whole-genome DNA microarrays. Strong Rec12 binding coincided with previously identified DSBs at the recombination hotspots ura4A, mbs1, and mbs2 and correlated with DSB formation at a new site. In addition, Rec12 binding corresponded to eight novel conversion hotspots and correlated with crossover density in segments of chromosome I. Notably, Rec12 binding inversely correlated with guanine-cytosine (GC) content, contrary to findings in Saccharomyces cerevisiae. Although both replication origins and Rec12-binding sites preferred AT-rich gene-free regions, they seemed to exclude each other. We also uncovered a connection between binding sites of Rec12 and meiotic cohesin Rec8. Rec12-binding peaks lay often within 2.5 kb of a Rec8-binding peak. Rec12 binding showed preference for large intergenic regions and was found to bind preferentially near to genes expressed strongly in meiosis. Surprisingly, Rec12 binding was also detected in centromeric core regions, which raises the intriguing possibility that Rec12 plays additional roles in meiotic chromosome dynamics.

  14. C. elegans HIM-17 links chromatin modification and competence for initiation of meiotic recombination.

    PubMed

    Reddy, Kirthi C; Villeneuve, Anne M

    2004-08-20

    Initiation of meiotic recombination by double-strand breaks (DSBs) must occur in a controlled fashion to avoid jeopardizing genome integrity. Here, we identify chromatin-associated protein HIM-17 as a link between chromatin state and DSB formation during C. elegans meiosis. Dependencies of several meiotic prophase events on HIM-17 parallel those seen for DSB-generating enzyme SPO-11: HIM-17 is essential for DSB formation but dispensable for homolog synapsis. Crossovers and chiasmata are eliminated in him-17 null mutants but are restored by artificially induced DSBs, indicating that all components required to convert DSBs into chiasmata are present. Unlike SPO-11, HIM-17 is also required for proper accumulation of histone H3 methylation at lysine 9 on meiotic prophase chromosomes. HIM-17 shares structural features with three proteins that interact genetically with LIN-35/Rb, a known component of chromatin-modifying complexes. Furthermore, DSB levels and incidence of chiasmata can be modulated by loss of LIN-35/Rb. These and other data suggest that chromatin state governs the timing of DSB competence.

  15. Numerical and spatial patterning of yeast meiotic DNA breaks by Tel1.

    PubMed

    Mohibullah, Neeman; Keeney, Scott

    2017-02-01

    The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and distribution. Mechanisms of this regulation remain poorly understood. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to reveal aspects of the contribution of the Saccharomyces cerevisiae DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM). A tel1Δ mutant has globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tel1 mutation similarly increases Spo11-oligonucleotide levels but mutating known Tel1 phosphotargets on Hop1 and Rec114 does not, implicating Tel1 kinase activity and clarifying roles of Tel1 phosphorylation substrates. Deep sequencing of Spo11 oligonucleotides demonstrates that Tel1 shapes the genome-wide DSB landscape in unexpected ways. Early in meiosis, Tel1 absence causes widespread changes in DSB distributions across large chromosomal domains. Many of these changes are erased as meiosis proceeds, however, illustrating homeostatic behavior of DSB regulatory systems. We further find that effects of Tel1 are distinct but partially overlapping with previously described contributions of the recombination regulator Cst9 (also known as Zip3). Finally, we provide evidence indicating that Tel1-dependent DSB interference influences the population-average DSB landscape but also demonstrate that locally inhibitory effects of an artificial hotspot insertion can be both Tel1-independent and chromosomal context-dependent. Our findings delineate Tel1 roles in regulating number and location of DSBs and illuminate the complex interplay between Tel1 and other pathways for DSB control.

  16. Phosphorylation of cohesin Rec11/SA3 by casein kinase 1 promotes homologous recombination by assembling the meiotic chromosome axis.

    PubMed

    Sakuno, Takeshi; Watanabe, Yoshinori

    2015-01-26

    In meiosis, cohesin is required for sister chromatid cohesion, as well as meiotic chromosome axis assembly and recombination. However, mechanisms underlying the multifunctional nature of cohesin remain elusive. Here, we show that fission yeast casein kinase 1 (CK1) plays a crucial role in assembling the meiotic chromosome axis (so-called linear element: LinE) and promoting recombination. An in vitro phosphorylation screening assay identified meiotic cohesin subunit Rec11/SA3 as an excellent substrate of CK1. The phosphorylation of Rec11 by CK1 mediates the interaction with the Rec10/Red1/SCP2 axis component, a key step in meiotic chromosome axis assembly, and is dispensable for sister chromatid cohesion. Crucially, the expression of Rec11-Rec10 fusion protein nearly completely bypasses the requirement for CK1 or cohesin phosphorylation for LinE assembly and recombination. This study uncovers a central mechanism of the cohesin-dependent assembly of the meiotic chromosome axis and recombination apparatus that acts independently of sister chromatid cohesion.

  17. Position- and orientation-independent activity of the Schizosaccharomyces pombe meiotic recombination hot spot M26

    PubMed Central

    Fox, Mary E.; Virgin, Jeffrey B.; Metzger, Jens; Smith, Gerald R.

    1997-01-01

    The activity of the M26 meiotic recombination hot spot of Schizosaccharomyces pombe depends on the presence of the heptamer 5′-ATGACGT-3′. Transplacement of DNA fragments containing the ade6-M26 gene to other chromosomal loci has previously demonstrated that the heptamer functions in some, but not all, transplacements, suggesting that hot spot activity depends on chromosomal context. In this study, hot spot activity was tested in the absence of gross DNA changes by using site-directed mutagenesis to create the heptamer sequence at novel locations in the genome. When created by mutagenesis of 1–4 bp in the ade6 and ura4 genes, the heptamer was active as a recombination hot spot, in an orientation-independent manner, at all locations tested. Thus, the heptamer sequence can create an active hot spot in other chromosomal contexts, provided that the gross chromosomal structure is not altered; this result is consistent with the hypothesis that a specific higher-order chromatin structure is required for M26 hot spot activity. PMID:9207111

  18. Drive against hotspot motifs in primates implicates the PRDM9 gene in meiotic recombination.

    PubMed

    Myers, Simon; Bowden, Rory; Tumian, Afidalina; Bontrop, Ronald E; Freeman, Colin; MacFie, Tammie S; McVean, Gil; Donnelly, Peter

    2010-02-12

    Although present in both humans and chimpanzees, recombination hotspots, at which meiotic crossover events cluster, differ markedly in their genomic location between the species. We report that a 13-base pair sequence motif previously associated with the activity of 40% of human hotspots does not function in chimpanzees and is being removed by self-destructive drive in the human lineage. Multiple lines of evidence suggest that the rapidly evolving zinc-finger protein PRDM9 binds to this motif and that sequence changes in the protein may be responsible for hotspot differences between species. The involvement of PRDM9, which causes histone H3 lysine 4 trimethylation, implies that there is a common mechanism for recombination hotspots in eukaryotes but raises questions about what forces have driven such rapid change.

  19. Matefin/SUN-1 phosphorylation is part of a surveillance mechanism to coordinate chromosome synapsis and recombination with meiotic progression and chromosome movement.

    PubMed

    Woglar, Alexander; Daryabeigi, Anahita; Adamo, Adele; Habacher, Cornelia; Machacek, Thomas; La Volpe, Adriana; Jantsch, Verena

    2013-01-01

    Faithful chromosome segregation during meiosis I depends on the establishment of a crossover between homologous chromosomes. This requires induction of DNA double-strand breaks (DSBs), alignment of homologs, homolog association by synapsis, and repair of DSBs via homologous recombination. The success of these events requires coordination between chromosomal events and meiotic progression. The conserved SUN/KASH nuclear envelope bridge establishes transient linkages between chromosome ends and cytoskeletal forces during meiosis. In Caenorhabditis elegans, this bridge is essential for bringing homologs together and preventing nonhomologous synapsis. Chromosome movement takes place during synapsis and recombination. Concomitant with the onset of chromosome movement, SUN-1 clusters at chromosome ends associated with the nuclear envelope, and it is phosphorylated in a chk-2- and plk-2-dependent manner. Identification of all SUN-1 phosphomodifications at its nuclear N terminus allowed us to address their role in prophase I. Failures in recombination and synapsis led to persistent phosphorylations, which are required to elicit a delay in progression. Unfinished meiotic tasks elicited sustained recruitment of PLK-2 to chromosome ends in a SUN-1 phosphorylation-dependent manner that is required for continued chromosome movement and characteristic of a zygotene arrest. Furthermore, SUN-1 phosphorylation supported efficient synapsis. We propose that signals emanating from a failure to successfully finish meiotic tasks are integrated at the nuclear periphery to regulate chromosome end-led movement and meiotic progression. The single unsynapsed X chromosome in male meiosis is precluded from inducing a progression delay, and we found it was devoid of a population of phosphorylated SUN-1. This suggests that SUN-1 phosphorylation is critical to delaying meiosis in response to perturbed synapsis. SUN-1 may be an integral part of a checkpoint system to monitor establishment of the

  20. Evidence that meiotic pairing starts at the telomeres: Molecular analysis of recombination in a family with a pericentric X chromosome inversion

    SciTech Connect

    Shashi, V.; Allinson, P.S.; Golden, W.L.; Kelly, T.E.

    1994-09-01

    Recent studies in yeast have shown that telomeres rather than centromeres lead in chromosome movement just prior to meiosis and may have a role in recombination. Cytological studies of meiosis in Drosophila and mice have shown that in pericentric inversion heterozygotes there is lack of loop formation, with recobmination seen only outside the inversion. In a family with Duchenne muscular dystrophy (DMD) we recognized that only affected males and carrier females had a pericentric X chromosome inversion (inv X(p11.4;q26)). Since the short arm inversion breakpoint was proximal to the DMD locus, it could not be implicated in the mutational event causing DMD. There was no history of infertility, recurrent miscarriages or liveborn unbalanced females to suggest there was recombination within the inversion. We studied 22 members over three generations to understand the pattern of meiotic recombination between the normal and the inverted X chromosome. In total, 17 meioses involving the inverted X chromosome in females were studied by cytogenetic analysis and 16 CA repeat polymorphisms along the length of the X chromosome. Results: (a) There was complete concordance between the segregation of the DMD mutation and the inverted X chromosome. (b) On DNA analysis, there was complete absence of recombination within the inverted segment. We also found no recombination at the DMD locus. Recombination was seen only at Xp22 and Xq27-28. (c) Recombination was seen in the same individual at both Xp22 and Xq27-28 without recombination otherwise. Conclusions: (1) Pericentric X inversions reduce the genetic map length of the chromosome, with the physical map length being normal. (2) Meiotic X chromosome pairing in this family is initiated at the telomeres. (3) Following telomeric pairing in pericentric X chromosome inversions, there is inhibition of recombination within the inversion and adjacent regions.

  1. Vilya, a component of the recombination nodule, is required for meiotic double-strand break formation in Drosophila.

    PubMed

    Lake, Cathleen M; Nielsen, Rachel J; Guo, Fengli; Unruh, Jay R; Slaughter, Brian D; Hawley, R Scott

    2015-10-09

    Meiotic recombination begins with the induction of programmed double-strand breaks (DSBs). In most organisms only a fraction of DSBs become crossovers. Here we report a novel meiotic gene, vilya, which encodes a protein with homology to Zip3-like proteins shown to determine DSB fate in other organisms. Vilya is required for meiotic DSB formation, perhaps as a consequence of its interaction with the DSB accessory protein Mei-P22, and localizes to those DSB sites that will mature into crossovers. In early pachytene Vilya localizes along the central region of the synaptonemal complex and to discrete foci. The accumulation of Vilya at foci is dependent on DSB formation. Immuno-electron microscopy demonstrates that Vilya is a component of recombination nodules, which mark the sites of crossover formation. Thus Vilya links the mechanism of DSB formation to either the selection of those DSBs that will become crossovers or to the actual process of crossing over.

  2. Acetylated Histone H3K9 is associated with meiotic recombination hotspots, and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast.

    PubMed

    Yamada, Shintaro; Ohta, Kunihiro; Yamada, Takatomi

    2013-04-01

    Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast.

  3. Acetylated Histone H3K9 is associated with meiotic recombination hotspots, and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast

    PubMed Central

    Yamada, Shintaro; Ohta, Kunihiro; Yamada, Takatomi

    2013-01-01

    Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast. PMID:23382177

  4. DNA recombination: the replication connection.

    PubMed

    Haber, J E

    1999-07-01

    Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.

  5. Meiotic recombination between yeast artificial chromosomes yields a single clone containing the entire BCL2 protooncogene.

    PubMed Central

    Silverman, G A; Green, E D; Young, R L; Jockel, J I; Domer, P H; Korsmeyer, S J

    1990-01-01

    The common translocation found in human follicular lymphoma, t(14;18)(q32;q21), results in deregulation of the BCL2 protoonocogene. The isolation of the intact gene would provide an essential substrate to analyze the molecular basis of this malignancy. Pulsed-field gel electrophoresis suggested that this three-exon gene was several hundred kilobases (kb) long. Therefore, a library of yeast artificial chromosome (YAC) clones was screened to isolate the intact BCL2 gene. Two clones, yA85B6 (200 kb) and yB206A6 (700 kb), were isolated by using polymerase chain reaction (PCR) assays specific for exon I/II and exon III, respectively. However, neither YAC contained the entire BCL2 locus. Since the two YACs were found to overlap by 60 kb, we sought to take advantage of the high recombination frequency in yeast and induce physical recombination between the two clones. Cells containing each YAC were mated and induced to undergo meiotic division and sporulation. Analysis of the resulting tetrads revealed a spore containing a single recombinant YAC of 800 kb. PCR assays and Southern blotting demonstrated that this recombined YAC contained the entire approximately 230-kb BCL2 gene. Furthermore, probe order was conserved and there was no evidence of overt rearrangements or deletions. These results indicate the feasibility of reconstructing large genomic segments with overlapping YAC clones to study genes spanning hundreds of kilobases. Images PMID:2263642

  6. Meiotic recombination analyses of individual chromosomes in male domestic pigs (Sus scrofa domestica).

    PubMed

    Mary, Nicolas; Barasc, Harmonie; Ferchaud, Stéphane; Billon, Yvon; Meslier, Frédéric; Robelin, David; Calgaro, Anne; Loustau-Dudez, Anne-Marie; Bonnet, Nathalie; Yerle, Martine; Acloque, Hervé; Ducos, Alain; Pinton, Alain

    2014-01-01

    For the first time in the domestic pig, meiotic recombination along the 18 porcine autosomes was directly studied by immunolocalization of MLH1 protein. In total, 7,848 synaptonemal complexes from 436 spermatocytes were analyzed, and 13,969 recombination sites were mapped. Individual chromosomes for 113 of the 436 cells (representing 2,034 synaptonemal complexes) were identified by immunostaining and fluorescence in situ hybridization (FISH). The average total length of autosomal synaptonemal complexes per cell was 190.3 µm, with 32.0 recombination sites (crossovers), on average, per cell. The number of crossovers and the lengths of the autosomal synaptonemal complexes showed significant intra- (i.e. between cells) and inter-individual variations. The distributions of recombination sites within each chromosomal category were similar: crossovers in metacentric and submetacentric chromosomes were concentrated in the telomeric regions of the p- and q-arms, whereas two hotspots were located near the centromere and in the telomeric region of acrocentrics. Lack of MLH1 foci was mainly observed in the smaller chromosomes, particularly chromosome 18 (SSC18) and the sex chromosomes. All autosomes displayed positive interference, with a large variability between the chromosomes.

  7. The synaptonemal complex has liquid crystalline properties and spatially regulates meiotic recombination factors

    PubMed Central

    Rog, Ofer; Köhler, Simone; Dernburg, Abby F

    2017-01-01

    The synaptonemal complex (SC) is a polymer that spans ~100 nm between paired homologous chromosomes during meiosis. Its striated, periodic appearance in electron micrographs led to the idea that transverse filaments within this structure ‘crosslink’ the axes of homologous chromosomes, stabilizing their pairing. SC proteins can also form polycomplexes, three-dimensional lattices that recapitulate the periodic structure of SCs but do not associate with chromosomes. Here we provide evidence that SCs and polycomplexes contain mobile subunits and that their assembly is promoted by weak hydrophobic interactions, indicative of a liquid crystalline phase. We further show that in the absence of recombination intermediates, polycomplexes recapitulate the dynamic localization of pro-crossover factors during meiotic progression, revealing how the SC might act as a conduit to regulate chromosome-wide crossover distribution. Properties unique to liquid crystals likely enable long-range signal transduction along meiotic chromosomes and underlie the rapid evolution of SC proteins. DOI: http://dx.doi.org/10.7554/eLife.21455.001 PMID:28045371

  8. Recombinant DNA: History of the Controversy.

    ERIC Educational Resources Information Center

    Vigue, Charles L.; Stanziale, William G.

    1979-01-01

    The hazards associated with recombinant DNA research are presented along with some social implications and the development of recombinant DNA research guidelines by the National Institutes of Health. (SA)

  9. Close, stable homolog juxtaposition during meiosis in budding yeast is dependent on meiotic recombination, occurs independently of synapsis, and is distinct from DSB-independent pairing contacts

    PubMed Central

    Peoples, Tamara L.; Dean, Eric; Gonzalez, Oscar; Lambourne, Lindsey; Burgess, Sean M.

    2002-01-01

    A site-specific recombination system that probes the relative probabilities that pairs of chromosomal loci collide with one another in living cells of budding yeast was used to explore the relative contributions of pairing, recombination, synaptonemal complex formation, and telomere clustering to the close juxtaposition of homologous chromosome pairs during meiosis. The level of Cre-mediated recombination between a pair of loxP sites located at an allelic position on homologous chromosomes was 13-fold greater than that between a pair of loxP sites located at ectopic positions on nonhomologous chromosomes. Mutations affecting meiotic recombination initiation and the processing of DNA double-strand breaks (DSBs) into single-end invasions (SEIs) reduced the levels of allelic Cre-mediated recombination levels by three- to sixfold. The severity of Cre/loxP phenotypes is presented in contrast to relatively weak DSB-independent pairing defects as assayed using fluorescence in situ hybridization for these mutants. Mutations affecting synaptonemal complex (SC) formation or crossover control gave wild-type levels of allelic Cre-mediated recombination. A delay in attaining maximum levels of allelic Cre-mediated recombination was observed for a mutant defective in telomere clustering. None of the mutants affected ectopic levels of recombination. These data suggest that stable, close homolog juxtaposition in yeast is distinct from pre-DSB pairing interactions, requires both DSB and SEI formation, but does not depend on crossovers or SC. PMID:12101126

  10. Meiotic recombination generates rich diversity in NK cell receptor genes, alleles, and haplotypes

    PubMed Central

    Norman, Paul J.; Abi-Rached, Laurent; Gendzekhadze, Ketevan; Hammond, John A.; Moesta, Achim K.; Sharma, Deepti; Graef, Thorsten; McQueen, Karina L.; Guethlein, Lisbeth A.; Carrington, Christine V.F.; Chandanayingyong, Dasdayanee; Chang, Yih-Hsin; Crespí, Catalina; Saruhan-Direskeneli, Güher; Hameed, Kamran; Kamkamidze, Giorgi; Koram, Kwadwo A.; Layrisse, Zulay; Matamoros, Nuria; Milà, Joan; Park, Myoung Hee; Pitchappan, Ramasamy M.; Ramdath, D. Dan; Shiau, Ming-Yuh; Stephens, Henry A.F.; Struik, Siske; Tyan, Dolly; Verity, David H.; Vaughan, Robert W.; Davis, Ronald W.; Fraser, Patricia A.; Riley, Eleanor M.; Ronaghi, Mostafa; Parham, Peter

    2009-01-01

    Natural killer (NK) cells contribute to the essential functions of innate immunity and reproduction. Various genes encode NK cell receptors that recognize the major histocompatibility complex (MHC) Class I molecules expressed by other cells. For primate NK cells, the killer-cell immunoglobulin-like receptors (KIR) are a variable and rapidly evolving family of MHC Class I receptors. Studied here is KIR3DL1/S1, which encodes receptors for highly polymorphic human HLA-A and -B and comprises three ancient allelic lineages that have been preserved by balancing selection throughout human evolution. While the 3DS1 lineage of activating receptors has been conserved, the two 3DL1 lineages of inhibitory receptors were diversified through inter-lineage recombination with each other and with 3DS1. Prominent targets for recombination were D0-domain polymorphisms, which modulate enhancer function, and dimorphism at position 283 in the D2 domain, which influences inhibitory function. In African populations, unequal crossing over between the 3DL1 and 3DL2 genes produced a deleted KIR haplotype in which the telomeric “half” was reduced to a single fusion gene with functional properties distinct from its 3DL1 and 3DL2 parents. Conversely, in Eurasian populations, duplication of the KIR3DL1/S1 locus by unequal crossing over has enabled individuals to carry and express alleles of all three KIR3DL1/S1 lineages. These results demonstrate how meiotic recombination combines with an ancient, preserved diversity to create new KIR phenotypes upon which natural selection acts. A consequence of such recombination is to blur the distinction between alleles and loci in the rapidly evolving human KIR gene family. PMID:19411600

  11. Variants of the protein PRDM9 differentially regulate a set of human meiotic recombination hotspots highly active in African populations.

    PubMed

    Berg, Ingrid L; Neumann, Rita; Sarbajna, Shriparna; Odenthal-Hesse, Linda; Butler, Nicola J; Jeffreys, Alec J

    2011-07-26

    PRDM9 is a major specifier of human meiotic recombination hotspots, probably via binding of its zinc-finger repeat array to a DNA sequence motif associated with hotspots. However, our view of PRDM9 regulation, in terms of motifs defined and hotspots studied, has a strong bias toward the PRDM9 A variant particularly common in Europeans. We show that population diversity can reveal a second class of hotspots specifically activated by PRDM9 variants common in Africans but rare in Europeans. These African-enhanced hotspots nevertheless share very similar properties with their counterparts activated by the A variant. The specificity of hotspot activation is such that individuals with differing PRDM9 genotypes, even within the same population, can use substantially if not completely different sets of hotspots. Each African-enhanced hotspot is activated by a distinct spectrum of PRDM9 variants, despite the fact that all are predicted to bind the same sequence motif. This differential activation points to complex interactions between the zinc-finger array and hotspots and identifies features of the array that might be important in controlling hotspot activity.

  12. Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice

    PubMed Central

    de Boer, Esther; Jasin, Maria; Keeney, Scott

    2015-01-01

    Meiotic recombination initiated by programmed double-strand breaks (DSBs) yields two types of interhomolog recombination products, crossovers and noncrossovers, but what determines whether a DSB will yield a crossover or noncrossover is not understood. In this study, we analyzed the influence of sex and chromosomal location on mammalian recombination outcomes by constructing fine-scale recombination maps in both males and females at two mouse hot spots located in different regions of the same chromosome. These include the most comprehensive maps of recombination hot spots in oocytes to date. One hot spot, located centrally on chromosome 1, behaved similarly in male and female meiosis: Crossovers and noncrossovers formed at comparable levels and ratios in both sexes. In contrast, at a distal hot spot, crossovers were recovered only in males even though noncrossovers were obtained at similar frequencies in both sexes. These findings reveal an example of extreme sex-specific bias in recombination outcome. We further found that estimates of relative DSB levels are surprisingly poor predictors of relative crossover frequencies between hot spots in males. Our results demonstrate that the outcome of mammalian meiotic recombination can be biased, that this bias can vary depending on location and cellular context, and that DSB frequency is not the only determinant of crossover frequency. PMID:26251527

  13. Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice.

    PubMed

    de Boer, Esther; Jasin, Maria; Keeney, Scott

    2015-08-15

    Meiotic recombination initiated by programmed double-strand breaks (DSBs) yields two types of interhomolog recombination products, crossovers and noncrossovers, but what determines whether a DSB will yield a crossover or noncrossover is not understood. In this study, we analyzed the influence of sex and chromosomal location on mammalian recombination outcomes by constructing fine-scale recombination maps in both males and females at two mouse hot spots located in different regions of the same chromosome. These include the most comprehensive maps of recombination hot spots in oocytes to date. One hot spot, located centrally on chromosome 1, behaved similarly in male and female meiosis: Crossovers and noncrossovers formed at comparable levels and ratios in both sexes. In contrast, at a distal hot spot, crossovers were recovered only in males even though noncrossovers were obtained at similar frequencies in both sexes. These findings reveal an example of extreme sex-specific bias in recombination outcome. We further found that estimates of relative DSB levels are surprisingly poor predictors of relative crossover frequencies between hot spots in males. Our results demonstrate that the outcome of mammalian meiotic recombination can be biased, that this bias can vary depending on location and cellular context, and that DSB frequency is not the only determinant of crossover frequency.

  14. The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo.

    PubMed

    Powers, Natalie R; Parvanov, Emil D; Baker, Christopher L; Walker, Michael; Petkov, Petko M; Paigen, Kenneth

    2016-06-01

    In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

  15. The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo

    PubMed Central

    Powers, Natalie R.; Parvanov, Emil D.; Baker, Christopher L.; Walker, Michael; Petkov, Petko M.; Paigen, Kenneth

    2016-01-01

    In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

  16. SPO11-independent DNA repair foci and their role in meiotic silencing.

    PubMed

    Carofiglio, Fabrizia; Inagaki, Akiko; de Vries, Sandra; Wassenaar, Evelyne; Schoenmakers, Sam; Vermeulen, Christie; van Cappellen, Wiggert A; Sleddens-Linkels, Esther; Grootegoed, J Anton; Te Riele, Hein P J; de Massy, Bernard; Baarends, Willy M

    2013-06-01

    In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11(YF/YF)), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.

  17. Recombinant DNA technology in apple.

    PubMed

    Gessler, Cesare; Patocchi, Andrea

    2007-01-01

    This review summarizes the achievements of almost 20 years of recombinant DNA technology applied to apple, grouping the research results into the sections: developing the technology, insect resistance, fungal disease resistance, self-incompatibility, herbicide resistance, fire blight resistance, fruit ripening, allergens, rooting ability, and acceptance and risk assessment. The diseases fire blight, caused by Erwinia amylovora, and scab, caused by Venturia inaequalis, were and still are the prime targets. Shelf life improvement and rooting ability of rootstocks are also relevant research areas. The tools to create genetically modified apples of added value to producers, consumers, and the environment are now available.

  18. Coordination of Recombination with Meiotic Progression in the Caenorhabditis elegans Germline by KIN-18, a TAO Kinase That Regulates the Timing of MPK-1 Signaling.

    PubMed

    Yin, Yizhi; Donlevy, Sean; Smolikove, Sarit

    2016-01-01

    Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase's role in germline development in higher eukaryotes.

  19. Decreased XY recombination and disturbed meiotic prophase I progression in an infertile 48, XYY, +sSMC man.

    PubMed

    Wang, Liu; Xu, Zhipeng; Iqbal, Furhan; Zhong, Liangwen; Zhang, Yuanwei; Wu, Caiyun; Zhou, Guixiang; Jiang, Hanwei; Bukhari, Ihtisham; Cooke, Howard J; Shi, Qinghua

    2015-06-01

    Small supernumerary marker chromosomes (sSMCs) are structurally abnormal rare chromosomes, difficult to characterize by karyotyping, and have been associated with minor dysmorphic features, azoospermia, and recurrent miscarriages. However, sSMC with a gonosomal trisomy has never been reported. Spermatocyte spreading and immunostaining were applied to detect meiotic prophase I progression, homologous chromosome pairing, synapsis, and recombination. In all the analyzed spermatocytes of the patient, the extra Y chromosome was not detected while the sSMC was present. The recombination frequency on autosomes was not affected, while the recombination frequencies on XY chromosome was significantly lower in the patient than in the controls. The meiotic prophase I progression was disturbed with significantly increased proportion of zygotene and decreased pachytene spermatocytes in the patients as compared with the controls. These findings highlight the importance of studies on meiotic behaviors in patients with an abnormal chromosomal constitution and provide an important framework for future studies, which may elucidate the impairment caused by sSMC in mammalian meiosis and fertility.

  20. Human Insulin from Recombinant DNA Technology

    NASA Astrophysics Data System (ADS)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  1. Correlations between Synaptic Initiation and Meiotic Recombination: A Study of Humans and Mice

    PubMed Central

    Gruhn, Jennifer R.; Al-Asmar, Nasser; Fasnacht, Rachael; Maylor-Hagen, Heather; Peinado, Vanessa; Rubio, Carmen; Broman, Karl W.; Hunt, Patricia A.; Hassold, Terry

    2016-01-01

    Meiotic recombination is initiated by programmed double strand breaks (DSBs), only a small subset of which are resolved into crossovers (COs). The mechanism determining the location of these COs is not well understood. Studies in plants, fungi, and insects indicate that the same genomic regions are involved in synaptic initiation and COs, suggesting that early homolog alignment is correlated with the eventual resolution of DSBs as COs. It is generally assumed that this relationship extends to mammals, but little effort has been made to test this idea. Accordingly, we conducted an analysis of synaptic initiation sites (SISs) and COs in human and mouse spermatocytes and oocytes. In contrast to our expectation, we observed remarkable sex- and species-specific differences, including pronounced differences between human males and females in both the number and chromosomal location of SISs. Further, the combined data from our studies in mice and humans suggest that the relationship between SISs and COs in mammals is a complex one that is not dictated by the sites of synaptic initiation as reported in other organisms, although it is clearly influenced by them. PMID:26749305

  2. PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans.

    PubMed

    Berg, Ingrid L; Neumann, Rita; Lam, Kwan-Wood G; Sarbajna, Shriparna; Odenthal-Hesse, Linda; May, Celia A; Jeffreys, Alec J

    2010-10-01

    PRDM9 has recently been identified as a likely trans regulator of meiotic recombination hot spots in humans and mice. PRDM9 contains a zinc finger array that, in humans, can recognize a short sequence motif associated with hot spots, with binding to this motif possibly triggering hot-spot activity via chromatin remodeling. We now report that human genetic variation at the PRDM9 locus has a strong effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Subtle changes within the zinc finger array can create hot-spot nonactivating or enhancing variants and can even trigger the appearance of a new hot spot, suggesting that PRDM9 is a major global regulator of hot spots in humans. Variation at the PRDM9 locus also influences aspects of genome instability-specifically, a megabase-scale rearrangement underlying two genomic disorders as well as minisatellite instability-implicating PRDM9 as a risk factor for some pathological genome rearrangements.

  3. Role of AtMSH7 in UV-B-induced DNA damage recognition and recombination.

    PubMed

    Lario, Luciana Daniela; Botta, Pablo; Casati, Paula; Spampinato, Claudia Patricia

    2015-06-01

    The mismatch repair (MMR) system maintains genome integrity by correcting replication-associated errors and inhibiting recombination between divergent DNA sequences. The basic features of the pathway have been highly conserved throughout evolution, although the nature and number of the proteins involved in this DNA repair system vary among organisms. Plants have an extra mismatch recognition protein, MutSγ, which is a heterodimer: MSH2-MSH7. To further understand the role of MSH7 in vivo, we present data from this protein in Arabidopsis thaliana. First, we generated transgenic plants that express β-glucuronidase (GUS) under the control of the MSH7 promoter. Histochemical staining of the transgenic plants indicated that MSH7 is preferentially expressed in proliferating tissues. Then, we identified msh7 T-DNA insertion mutants. Plants deficient in MSH7 show increased levels of UV-B-induced cyclobutane pyrimidine dimers relative to wild-type (WT) plants. Consistent with the patterns of MSH7 expression, we next analysed the role of the protein during somatic and meiotic recombination. The frequency of somatic recombination between homologous or homeologous repeats (divergence level of 1.6%) was monitored using a previously described GUS recombination reporter assay. Disruption of MSH7 has no effect on the rates of somatic homologous or homeologous recombination under control conditions or after UV-B exposure. However, the rate of meiotic recombination between two genetically linked seed-specific fluorescent markers was 97% higher in msh7 than in WT plants. Taken together, these results suggest that MSH7 is involved in UV-B-induced DNA damage recognition and in controlling meiotic recombination.

  4. Vilya, a component of the recombination nodule, is required for meiotic double-strand break formation in Drosophila

    PubMed Central

    Lake, Cathleen M; Nielsen, Rachel J; Guo, Fengli; Unruh, Jay R; Slaughter, Brian D; Hawley, R Scott

    2015-01-01

    Meiotic recombination begins with the induction of programmed double-strand breaks (DSBs). In most organisms only a fraction of DSBs become crossovers. Here we report a novel meiotic gene, vilya, which encodes a protein with homology to Zip3-like proteins shown to determine DSB fate in other organisms. Vilya is required for meiotic DSB formation, perhaps as a consequence of its interaction with the DSB accessory protein Mei-P22, and localizes to those DSB sites that will mature into crossovers. In early pachytene Vilya localizes along the central region of the synaptonemal complex and to discrete foci. The accumulation of Vilya at foci is dependent on DSB formation. Immuno-electron microscopy demonstrates that Vilya is a component of recombination nodules, which mark the sites of crossover formation. Thus Vilya links the mechanism of DSB formation to either the selection of those DSBs that will become crossovers or to the actual process of crossing over. DOI: http://dx.doi.org/10.7554/eLife.08287.001 PMID:26452093

  5. The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis.

    PubMed

    Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J Julian; Gartner, Anton

    2016-03-01

    Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis.

  6. Endogenous Small RNA Mediates Meiotic Silencing of a Novel DNA Transposon.

    PubMed

    Wang, Yizhou; Smith, Kristina M; Taylor, John W; Freitag, Michael; Stajich, Jason E

    2015-06-23

    Genome defense likely evolved to curtail the spread of transposable elements and invading viruses. A combination of effective defense mechanisms has been shown to limit colonization of the Neurospora crassa genome by transposable elements. A novel DNA transposon named Sly1-1 was discovered in the genome of the most widely used laboratory "wild-type" strain FGSC 2489 (OR74A). Meiotic silencing by unpaired DNA, also simply called meiotic silencing, prevents the expression of regions of the genome that are unpaired during karyogamy. This mechanism is posttranscriptional and is proposed to involve the production of small RNA, so-called masiRNAs, by proteins homologous to those involved in RNA interference-silencing pathways in animals, fungi, and plants. Here, we demonstrate production of small RNAs when Sly1-1 was unpaired in a cross between two wild-type strains. These small RNAs are dependent on SAD-1, an RNA-dependent RNA polymerase necessary for meiotic silencing. We present the first case of endogenously produced masiRNA from a novel N. crassa DNA transposable element.

  7. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

    1994-10-18

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

  8. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

    1994-01-01

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

  9. Variation in meiotic recombination frequencies between allelic transgenes inserted at different sites in the Drosophila melanogaster genome.

    PubMed

    McMahan, Susan; Kohl, Kathryn P; Sekelsky, Jeff

    2013-08-07

    Meiotic crossovers are distributed nonrandomly across the genome. Classic studies in Drosophila suggest that the position of a gene along a chromosome arm can affect the outcome of the recombination process, with proximity to the centromere being associated with lower crossing over. To examine this phenomenon molecularly, we developed an assay that measures meiotic crossovers and noncrossover gene conversions between allelic transgenes inserted into different genomic positions. To facilitate collecting a large number of virgin females, we developed a useful genetic system that kills males and undesired classes of females. We found that the recombination frequency at a site in the middle of the X chromosome, where crossovers are normally frequent, was similar to the frequency at the centromere-proximal end of the euchromatin, where crossovers are normally infrequent. In contrast, we recovered no recombinants--crossovers or noncrossovers--at a site on chromosome 4 and at a site toward the distal end of the X chromosome. These results suggest that local sequence or chromatin features have a stronger impact on recombination rates in this transgene assay than position along the chromosome arm.

  10. Complex relationship between meiotic recombination frequency and autosomal synaptonemal complex length per cell in normal human males.

    PubMed

    Pan, Zhenzhen; Yang, Qingling; Ye, Nan; Wang, Liu; Li, Jianhua; Yu, Dexin; Cooke, Howard J; Shi, Qinghua

    2012-03-01

    Although the relationship between meiotic recombination frequency and synaptonemal complex (SC) length has been of interest for a long time, how recombination frequency is related to SC length has not been carefully explored. To address this question, we have measured the meiotic recombination frequency as represented by MLH1 foci in 889 pachytene spermatocytes and measured the length of 19,558 autosomal SCs from 10 human males. A complex relationship between the number of MLH1 foci and total autosomal SC length per cell was observed. A positive correlation with significant correlation coefficients between the two variables was found in eight of the ten donors examined, with three donors showing weak correlation, and five showing moderate correlation. Two donors who did not show any correlation between the two variables were identified for the first time. Moreover, most cells with similar total autosomal SC length showed very different numbers of MLH1 foci both between individuals and even within an individual, and vice versa. Our data provide the first evidence for a complex relationship between the recombination frequency and total length of autosomal SCs per cell in human males.

  11. The meiotic stage of nondisjunction in trisomy 21: Determination by using DNA polymorphisms

    PubMed Central

    Antonarakis, Stylianos E.; Petersen, Michael B.; McInnis, Melvin G.; Adelsberger, Patricia A.; Schinzel, Albert A.; Binkert, Franz; Pangalos, Constantine; Raoul, Odile; Slaugenhaupt, Susan A.; Hafez, Mohamed; Cohen, Maimon M.; Roulson, Diane; Schwartz, Stuart; Mikkelsen, Margareta; Tranebjaerg, Lisbeth; Greenberg, Frank; Hoar, David I.; Rudd, Noreen L.; Warren, Andrew C.; Metaxotou, Caterina; Bartsocas, Christos; Chakravarti, Aravinda

    1992-01-01

    We have studied DNA polymorphisms at loci in the pericentromeric region on the long arm of chromosome 21 in 200 families with trisomy 21, in order to determine the meiotic origin of nondisjunction. Maintenance of heterozygosity for parental markers in the individual with trisomy 21 was interpreted as resulting from a meiosis I error, while reduction to homozygosity was attributed to a meiosis II error. Nondisjunction was paternal in 9 cases and was maternal in 188 cases, as reported earlier. Among the 188 maternal cases, nondisjunction occurred in meiosis I in 128 cases and in meiosis II in 38 cases; in 22 cases the DNA markers used were uninformative. Therefore meiosis I was responsible for 77.1% and meiosis II for 22.9% of maternal nondisjunction. Among the 9 paternal nondisjunction cases the error occurred in meiosis I in 2 cases (22.2%) and in meiosis II in 7 (77.8%) cases. Since there was no significant difference in the distribution of maternal ages between maternal I error versus maternal II error, it is unlikely that an error at a particular meiotic stage contributes significantly to the increasing incidence of Down syndrome with advancing maternal age. Although the DNA polymorphisms used were at loci which map close to the centromere, it is likely that rare errors in meiotic-origin assignments may have occurred because of a small number of crossovers between the markers and the centromere. Analysis of these polymorphisms may provide a more accurate understanding of the meiotic stage of nondisjunction in trisomy 21 than that previously provided by chromosomal heteromorphisms. ImagesFigure 1 PMID:1347192

  12. Strategies for RNA-Guided DNA Recombination

    NASA Astrophysics Data System (ADS)

    Angeleska, Angela; Jonoska, Nataša; Saito, Masahico; Landweber, Laura F.

    We present a model for homologous DNA recombination events guided by double-stranded RNA (dsRNA) templates, and apply this model to DNA rearrangements in some groups of ciliates, such as Stylonychia or Oxytricha. In these organisms, differentiation of a somatic macronucleus from a germline micronucleus involves extensive gene rearrangement, which can be modeled as topological braiding of the DNA, with the template-guided alignment proceeding through DNA branch migration. We show that a graph structure, which we refer to as an assembly graph, containing only 1- and 4-valent vertices can provide a physical representation of the DNA at the time of recombination. With this representation, 4-valent vertices correspond to the alignment of the recombination sites, and we model the actual recombination event as smoothing of these vertices.

  13. Disease diagnosis by recombinant DNA methods

    SciTech Connect

    Caskey, C.T.

    1987-06-05

    Recombinant DNA procedures have now been applied to the problem of the identification of molecular defects in man that account for heritable diseases, somatic mutations associated with neoplasia, and acquired infectious disease. Thus, recombinant DNA technology has rapidly expanded the ability to diagnose disease. Substantial advances in the simplification of procedures for diagnostic purposes have been made, and the informed physician has gained in diagnostic accuracy as a consequence of these developments. The wide application of recombinant DNA diagnostics will depend on simplicity, speed of results, and cost containment. 66 references, 7 figures.

  14. The dual role of HOP2 in mammalian meiotic homologous recombination

    PubMed Central

    Pezza, Roberto J.; Voloshin, Oleg N.; Volodin, Alexander A.; Boateng, Kingsley A.; Bellani, Marina A.; Mazin, Alexander V.; Camerini-Otero, R. Daniel

    2014-01-01

    Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2–MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1−/− spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2–MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis. PMID:24304900

  15. Cumulus Cells Block Oocyte Meiotic Resumption via Gap Junctions in Cumulus Oocyte Complexes Subjected to DNA Double-Strand Breaks.

    PubMed

    Sun, Ming-Hong; Zheng, Jie; Xie, Feng-Yun; Shen, Wei; Yin, Shen; Ma, Jun-Yu

    2015-01-01

    During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.

  16. Recombination in Eukaryotic Single Stranded DNA Viruses

    PubMed Central

    Martin, Darren P.; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

    2011-01-01

    Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution. PMID:21994803

  17. Recombination in eukaryotic single stranded DNA viruses.

    PubMed

    Martin, Darren P; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

    2011-09-01

    Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution.

  18. Bivalent Formation 1, a plant-conserved gene, encodes an OmpH/coiled-coil motif-containing protein required for meiotic recombination in rice.

    PubMed

    Zhou, Lian; Han, Jingluan; Chen, Yuanling; Wang, Yingxiang; Liu, Yao-Guang

    2017-03-24

    Meiosis is essential for eukaryotic sexual reproduction and plant fertility. In comparison with over 80 meiotic genes identified in Arabidopsis, there are only ~30 meiotic genes characterized in rice (Oryza sativa L.). Many genes involved in the regulation of meiotic progression remain to be determined. In this study, we identified a sterile rice mutant and cloned a new meiotic gene, OsBVF1 (Bivalent Formation 1) by map-based cloning. Molecular genetics and cytological approaches were carried out to address the function of OsBVF1 in meiosis. Phylogenetic analyses were used to study the evolution of OsBVF1 and its homologs in plant species. Here we showed that the bvf1 male meiocytes were defective in formation of meiotic double strand break, thereby resulting in a failure of bivalent formation in diakinesis and unequal chromosome segregation in anaphase I. The causal gene, OsBVF1, encodes a unique OmpH/coiled-coil motif-containing protein and its homologs are highly conserved in the plant kingdom and seem to be a single-copy gene in the majority of plant species. Our study demonstrates that OsBVF1 is a novel plant-conserved factor involved in meiotic recombination in rice, providing a new insight into understanding of meiotic progression regulation.

  19. The 14-kDa Dynein Light Chain-Family Protein Dlc1 Is Required for Regular Oscillatory Nuclear Movement and Efficient Recombination during Meiotic Prophase in Fission Yeast

    PubMed Central

    Miki, Futaba; Okazaki, Koei; Shimanuki, Mizuki; Yamamoto, Ayumu; Hiraoka, Yasushi; Niwa, Osami

    2002-01-01

    A Schizosaccharomyces pombe spindle pole body (SPB) protein interacts in a two-hybrid system with Dlc1, which belongs to the 14-kDa Tctex-1 dynein light chain family. Green fluorescent protein-tagged Dlc1 accumulated at the SPB throughout the life cycle. During meiotic prophase, Dlc1 was present along astral microtubules and microtubule-anchoring sites on the cell cortex, reminiscent of the cytoplasmic dynein heavy chain Dhc1. In a dlc1-null mutant, Dhc1-dependent nuclear movement in meiotic prophase became irregular in its duration and direction. Dhc1 protein was displaced from the cortex anchors and the formation of microtubule bundle(s) that guide nuclear movement was impaired in the mutant. Meiotic recombination in the dlc1 mutant was reduced to levels similar to that in the dhc1 mutant. Dlc1 and Dhc1 also have roles in karyogamy and rDNA relocation during the sexual phase. Strains mutated in both the dlc1 and dhc1 loci displayed more severe defects in recombination, karyogamy, and sporulation than in either single mutant alone, suggesting that Dlc1 is involved in nuclear events that are independent of Dhc1. S. pombe contains a homolog of the 8-kDa dynein light chain, Dlc2. This class of dynein light chain, however, is not essential in either the vegetative or sexual phases. PMID:11907273

  20. Single-molecule observation of DNA compaction by meiotic protein SYCP3

    PubMed Central

    Syrjänen, Johanna L; Heller, Iddo; Candelli, Andrea; Davies, Owen R; Peterman, Erwin J G; Wuite, Gijs J L; Pellegrini, Luca

    2017-01-01

    In a previous paper (Syrjänen et al., 2014), we reported the first structural characterisation of a synaptonemal complex (SC) protein, SYCP3, which led us to propose a model for its role in chromosome compaction during meiosis. As a component of the SC lateral element, SYCP3 has a critical role in defining the specific chromosome architecture required for correct meiotic progression. In the model, the reported compaction of chromosomal DNA caused by SYCP3 would result from its ability to bridge distant sites on a DNA molecule with the DNA-binding domains located at each end of its strut-like structure. Here, we describe a single-molecule assay based on optical tweezers, fluorescence microscopy and microfluidics that, in combination with bulk biochemical data, provides direct visual evidence for our proposed mechanism of SYCP3-mediated chromosome organisation. DOI: http://dx.doi.org/10.7554/eLife.22582.001 PMID:28287952

  1. Chromosome pairing and meiotic recombination in Neurospora crassa spo11 mutants.

    PubMed

    Bowring, Frederick J; Yeadon, P Jane; Stainer, Russell G; Catcheside, David E A

    2006-08-01

    Some organisms, such as mammals, green plants and fungi, require double-strand breaks in DNA (DSBs) for synapsis of homologous chromosomes at pachynema. Drosophila melanogaster and Caenorhabditis elegans are exceptions, achieving synapsis independently of DSB. SPO11 is responsible for generating DSBs and perhaps for the initiation of recombination in all organisms. Although it was previously suggested that Neurospora may not require DSBs for synapsis, we report here that mutation of Neurospora spo11 disrupts meiosis, abolishing synapsis of homologous chromosomes during pachynema and resulting in ascospores that are frequently aneuploid and rarely viable. Alignment of homologues is partially restored after exposure of spo11 perithecia to ionising radiation. Crossing over in a spo11 mutant is reduced in two regions of the Neurospora genome as expected, but is unaffected in a third.

  2. Mcp7, a meiosis-specific coiled-coil protein of fission yeast, associates with Meu13 and is required for meiotic recombination

    PubMed Central

    Saito, Takamune T.; Tougan, Takahiro; Kasama, Takashi; Okuzaki, Daisuke; Nojima, Hiroshi

    2004-01-01

    We previously showed that Meu13 of Schizosaccharomyces pombe functions in homologous pairing and recombination at meiosis I. Here we show that a meiosis-specific gene encodes a coiled-coil protein that complexes with Meu13 during meiosis in vivo. This gene denoted as mcp7+ (after meiotic coiled-coil protein) is an ortholog of Mnd1 of Saccharomyces cerevisiae. Mcp7 proteins are detected on meiotic chromatin. The phenotypes of mcp7Δ cells are similar to those of meu13Δ cells as they show reduced recombination rates and spore viability and produce spores with abnormal morphology. However, a delay in initiation of meiosis I chromosome segregation of mcp7Δ cells is not so conspicuous as meu13Δ cells, and no meiotic delay is observed in mcp7Δmeu13Δ cells. Mcp7 and Meu13 proteins depend on each other differently; Mcp7 becomes more stable in meu13Δ cells, whereas Meu13 becomes less stable in mcp7Δ cells. Genetic analysis shows that Mcp7 acts in the downstream of Dmc1, homologs of Escherichia coli RecA protein, for both recombination and subsequent sporulation. Taken together, we conclude that Mcp7 associates with Meu13 and together they play a key role in meiotic recombination. PMID:15210864

  3. Mcp7, a meiosis-specific coiled-coil protein of fission yeast, associates with Meu13 and is required for meiotic recombination.

    PubMed

    Saito, Takamune T; Tougan, Takahiro; Kasama, Takashi; Okuzaki, Daisuke; Nojima, Hiroshi

    2004-01-01

    We previously showed that Meu13 of Schizosaccharomyces pombe functions in homologous pairing and recombination at meiosis I. Here we show that a meiosis-specific gene encodes a coiled-coil protein that complexes with Meu13 during meiosis in vivo. This gene denoted as mcp7+ (after meiotic coiled-coil protein) is an ortholog of Mnd1 of Saccharomyces cerevisiae. Mcp7 proteins are detected on meiotic chromatin. The phenotypes of mcp7Delta cells are similar to those of meu13Delta cells as they show reduced recombination rates and spore viability and produce spores with abnormal morphology. However, a delay in initiation of meiosis I chromosome segregation of mcp7Delta cells is not so conspicuous as meu13Delta cells, and no meiotic delay is observed in mcp7Deltameu13Delta cells. Mcp7 and Meu13 proteins depend on each other differently; Mcp7 becomes more stable in meu13Delta cells, whereas Meu13 becomes less stable in mcp7Delta cells. Genetic analysis shows that Mcp7 acts in the downstream of Dmc1, homologs of Escherichia coli RecA protein, for both recombination and subsequent sporulation. Taken together, we conclude that Mcp7 associates with Meu13 and together they play a key role in meiotic recombination.

  4. Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

    PubMed

    Lyndaker, Amy M; Lim, Pei Xin; Mleczko, Joanna M; Diggins, Catherine E; Holloway, J Kim; Holmes, Rebecca J; Kan, Rui; Schlafer, Donald H; Freire, Raimundo; Cohen, Paula E; Weiss, Robert S

    2013-01-01

    The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

  5. DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts.

    PubMed Central

    Murakami, H; Nurse, P

    2000-01-01

    The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast. PMID:10861204

  6. The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA.

    PubMed

    Decker, Logan M; Xiao, Hua; Boone, Erin C; Vierling, Michael M; Shanker, Benjamin S; Kingston, Shanika L; Boone, Shannon F; Haynes, Jackson B; Shiu, Patrick K T

    2017-02-07

    In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus to combat these repetitive elements. One of these defense mechanisms is known as meiotic silencing by unpaired DNA (MSUD), which is an RNA silencing system that identifies and silences unpaired genes during meiosis. Utilizing common RNAi proteins such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified another silencing component known as the cap-binding complex (CBC). Made up of CBP20 and CBP80 (cap-binding proteins 20 and 80), CBC associates with the 5' cap of nascent mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. Similar to animals but unlike plants, CBP20's robust nuclear re-entry is shown to be dependent on CBP80. CBC interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), directly linking the two cellular factors.

  7. The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA

    PubMed Central

    Decker, Logan M.; Xiao, Hua; Boone, Erin C.; Vierling, Michael M.; Shanker, Benjamin S.; Kingston, Shanika L.; Boone, Shannon F.; Haynes, Jackson B.; Shiu, Patrick K.T.

    2017-01-01

    In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. One of these defense mechanisms is called meiotic silencing by unpaired DNA (MSUD), which identifies and silences unpaired genes during meiosis. Utilizing common RNA interference (RNAi) proteins, such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified an additional silencing component, namely the cap-binding complex (CBC). Made up of cap-binding proteins CBP20 and CBP80, CBC associates with the 5′ cap of mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. As seen in animals but not in plants, CBP20’s robust nuclear import depends on CBP80 in Neurospora. CBC interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), directly linking the two cellular factors. PMID:28179391

  8. Interpopulation hybridization generates meiotically stable rDNA epigenetic variants in allotetraploid Tragopogon mirus.

    PubMed

    Matyášek, Roman; Dobešová, Eva; Húska, Dalibor; Ježková, Ivana; Soltis, Pamela S; Soltis, Douglas E; Kovařík, Aleš

    2016-02-01

    Uniparental silencing of 35S rRNA genes (rDNA), known as nucleolar dominance (ND), is common in interspecific hybrids. Allotetraploid Tragopogon mirus composed of Tragopogon dubius (d) and Tragopogon porrifolius (p) genomes shows highly variable ND. To examine the molecular basis of such variation, we studied the genetic and epigenetic features of rDNA homeologs in several lines derived from recently and independently formed natural populations. Inbred lines derived from T. mirus with a dominant d-rDNA homeolog transmitted this expression pattern over generations, which may explain why it is prevalent among natural populations. In contrast, lines derived from the p-rDNA dominant progenitor were meiotically unstable, frequently switching to co-dominance. Interpopulation crosses between progenitors displaying reciprocal ND resulted in d-rDNA dominance, indicating immediate suppression of p-homeologs in F1 hybrids. Original p-rDNA dominance was not restored in later generations, even in those segregants that inherited the corresponding parental rDNA genotype, thus indicating the generation of additional p-rDNA and d-rDNA epigenetic variants. Despite preserved intergenic spacer (IGS) structure, they showed altered cytosine methylation and chromatin condensation patterns, and a correlation between expression, hypomethylation of RNA Pol I promoters and chromatin decondensation was apparent. Reversion of such epigenetic variants occurred rarely, resulting in co-dominance maintained in individuals with distinct genotypes. Generally, interpopulation crosses may generate epialleles that are not present in natural populations, underlying epigenetic dynamics in young allopolyploids. We hypothesize that highly expressed variants with distinct IGS features may induce heritable epigenetic reprogramming of the partner rDNA arrays, harmonizing the expression of thousands of genes in allopolyploids.

  9. DNA Sequence Alignment during Homologous Recombination.

    PubMed

    Greene, Eric C

    2016-05-27

    Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination.

  10. Genetics of meiosis and recombination in mice.

    PubMed

    Bolcun-Filas, Ewelina; Schimenti, John C

    2012-01-01

    Meiosis is one of the most critical developmental processes in sexually reproducing organisms. One round of DNA replication followed by two rounds of cell divisions results in generation of haploid gametes (sperm and eggs in mammals). Meiotic failure typically leads to infertility in mammals. In the process of meiotic recombination, maternal and paternal genomes are shuffled, creating new allelic combinations and thus genetic variety. However, in order to achieve this, meiotic cells must self-inflict DNA damage in the form of programmed double-strand breaks (DSBs). Complex processes evolved to ensure proper DSB repair, and to do so in a way that favors interhomolog reciprocal recombination and crossovers. The hallmark of meiosis, a structurally conserved proteinaceous structure called the synaptonemal complex, is found only in meiotic cells. Conversely, meiotic homologous recombination is an adaptation of the mitotic DNA repair process but involving specialized proteins. In this chapter, we summarize current developments in mammalian meiosis enabled by genetically modified mice.

  11. A new light on the meiotic DSB catalytic complex.

    PubMed

    Robert, Thomas; Vrielynck, Nathalie; Mézard, Christine; de Massy, Bernard; Grelon, Mathilde

    2016-06-01

    Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs). More than 15 years ago, Spo11 was identified as the protein responsible for meiotic DSB formation, notably because of its striking similarities with the A subunit of topoisomerase VI (TopoVI). TopoVI are enzymes that modify DNA topology by generating transient DSBs and are active as heterotetramers, composed of two A and two B subunits. A2 dimers catalyse the DNA cleavage reaction, whereas the B subunits regulate A2 conformation, DNA capture, cleavage and re-ligation. The recent identification in plants and mammals of a B-like TopoVI subunit that interacts with SPO11 and is required for meiotic DSB formation makes us to reconsider our understanding of the meiotic DSB catalytic complex. We provide here an overview of the knowledge on TopoVI structure and mode of action and we compare them with their meiotic counterparts. This allows us to discuss the nature, structure and functions of the meiotic TopoVI-like complex during meiotic DSB formation.

  12. Transcription and Recombination: When RNA Meets DNA

    PubMed Central

    Aguilera, Andrés; Gaillard, Hélène

    2014-01-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. PMID:25085910

  13. Recombinant DNA: Scientific and Social Perspectives.

    ERIC Educational Resources Information Center

    Vandegrift, Vaughn

    1979-01-01

    This article is designed to inform chemical educators not engaged in this technology as to the nature and methods used in the technology, the reasons for scientific and social concern, and the attempts made to assuage concerns involving recombinant DNA research. (author/BB)

  14. Vaccine development using recombinant DNA technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vaccines induce an immune response in the host that subsequently recognizes infectious agents and helps fight off the disease; vaccines must do this without causing the disease. This paper reviews the development of recombinant DNA technologies as a means of providing new ways for attenuating diseas...

  15. Science: The Recombinant DNA Advisory Committee.

    ERIC Educational Resources Information Center

    Wright, Susan

    1979-01-01

    Reports on the status of the Recombinant DNA Advisory Committee (RAC) and attempts to rationalize Suburban Highway Policy. Effective communication among members of the RAC is a current problem facing the committee. A federal transportation priority spending policy is suggested during these times of money and fuel shortages. (MA)

  16. Enhancement of spontaneous mitotic recombination by the meiotic mutant spo11-1 in Saccharomyces cerevisiae

    SciTech Connect

    Bruschi, C.V.; Esposito, M.S.

    1983-12-01

    Both nonreciprocal and reciprocal mitotic recombination are enhanced by the recessive mutant spo11-1, which was previously shown to affect meiosis by decreasing recombination and increasing nondisjunction. The mitotic effects are not distributed equally in all chromosomal regions. The genotypes of mitotic recombinants in spo11-1/spo11-1 diploid cells provide further evidence that widely spaced chromosomal markers undergo coincident conversion in mitosis.

  17. Three Additional Linkage Groups That Repress Transcription and Meiotic Recombination in the Mating-Type Region of Schizosaccharomyces Pombe

    PubMed Central

    Thon, G.; Cohen, A.; Klar, A. J.

    1994-01-01

    The mating-type genes of Schizosaccharomyces pombe are found at three locations in the same chromosomal region. These genes are in an active configuration at the mat1 locus and in an inactive configuration at the mat2 and mat3 loci. The mechanism that represses transcription of mat2 and mat3 also inactivates other promoters introduced nearby and is accompanied by a block to meiotic recombination in the mat2-mat3 interval, suggesting that this mechanism involves a particular chromatin structure. We present evidence that the transcription and recombination blocks require three newly defined trans-acting loci, clr2, clr3 and clr4, in addition to the previously identified clr1, rik1 and swi6 loci. We also investigated the role of mat2 cis-acting sequences in silencing. Four cis-acting elements that repress mat2 in a plasmid context were previously identified. Deletion of two of these elements proved to have little effect in a chromosomal context. However, when combined with mutations in trans-acting genes, deletion of the same two elements greatly enhanced mat2 expression. The observed cumulative effects suggest a redundancy in the silencing mechanism. PMID:8001791

  18. mei-W68 in Drosophila melanogaster encodes a Spo11 homolog: evidence that the mechanism for initiating meiotic recombination is conserved.

    PubMed

    McKim, K S; Hayashi-Hagihara, A

    1998-09-15

    Meiotic recombination requires the action of several gene products in both Saccharomyces cerevisiae and Drosophila melanogaster. Genetic studies in D. melanogaster have shown that the mei-W68 gene is required for all meiotic gene conversion and crossing-over. We cloned mei-W68 using a new genetic mapping method in which P elements are used to promote crossing-over at their insertion sites. This resulted in the high-resolution mapping of mei-W68 to a <18-kb region that contains a homolog of the S. cerevisiae spo11 gene. Molecular analysis of several mutants confirmed that mei-W68 encodes an spo11 homolog. Spo11 and MEI-W68 are members of a family of proteins similar to a novel type II topoisomerase. On the basis of this and other lines of evidence, Spo11 has been proposed to be the enzymatic activity that creates the double-strand breaks needed to initiate meiotic recombination. This raises the possibility that recombination in Drosophila is also initiated by double-strand breaks. Although these homologous genes are required absolutely for recombination in both species, their roles differ in other respects. In contrast to spo11, mei-W68 is not required for synaptonemal complex formation and does have a mitotic role.

  19. Monitoring DNA recombination initiated by HO endonuclease.

    PubMed

    Sugawara, Neal; Haber, James E

    2012-01-01

    DNA double-strand breaks (DSBs) have proven to be very potent initiators of recombination in yeast and other organisms. A single, site-specific DSB initiates homologous DNA repair events such as gene conversion, break-induced replication, and single-strand annealing, as well as nonhomologous end joining, microhomology-mediated end joining, and new telomere addition. When repair is either delayed or prevented, a single DSB can trigger checkpoint-mediated cell cycle arrest. In budding yeast, expressing the HO endonuclease under the control of a galactose-inducible promoter has been instrumental in the study of these processes by providing us a way to synchronously induce a DSB at a unique site in vivo. We describe how the HO endonuclease has been used to study the recombination events in mating-type (MAT) switching. Southern blots provide an overview of the process by allowing one to examine the formation of the DSB, DNA degradation at the break, and formation of the product. Denaturing gels and slot blots as well as PCR have provided important tools to follow the progression of resection in wild-type and mutant cells. PCR has also been important in allowing us to follow the kinetics of certain recombination intermediates such as the initiation of repair DNA synthesis or the removal of nonhomologous Y sequences during MAT switching. Finally chromatin immunoprecipitation has been used to follow the recruitment of key proteins to the DSB and in subsequent steps in DSB repair.

  20. Meiotic Recombination in Somatic Cell Nuclear Transfer Bulls and Their Offspring

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In mammals, homologous chromosome pairing and recombination are essential events for meiosis. The generation of reciprocal exchanges of genetic material ensure both genetic diversity and the proper segregation of homologous chromosomes. With the advent of reproductive biotechnologies such as somat...

  1. Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis

    SciTech Connect

    Cole, G.M.; Mortimer, R.K. ); Schild, D. )

    1989-07-01

    The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/{alpha} cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of {beta}-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.

  2. Meiotic DNA double-strand break repair requires two nucleases, MRN and Ctp1, to produce a single size class of Rec12 (Spo11)-oligonucleotide complexes.

    PubMed

    Milman, Neta; Higuchi, Emily; Smith, Gerald R

    2009-11-01

    Programmed DNA double-strand breaks (DSBs) in meiosis are formed by Spo11 (Rec12 in fission yeast), a topoisomerase II-like protein, which becomes covalently attached to DNA 5' ends. For DSB repair through homologous recombination, the protein must be removed from these DNA ends. We show here that Rec12 is endonucleolytically removed from DSB ends attached to a short oligonucleotide (Rec12-oligonucleotide complex), as is Spo11 in budding yeast. Fission yeast, however, has only one size class of Rec12-oligonucleotide complexes, whereas budding yeast has two size classes, suggesting different endonucleolytic regulatory mechanisms. Rec12-oligonucleotide generation strictly requires Ctp1 (Sae2 nuclease homolog), the Rad32 (Mre11) nuclease domain, and Rad50 of the MRN complex. Surprisingly, Nbs1 is not strictly required, indicating separable roles for the MRN subunits. On the basis of these and other data, we propose that Rad32 nuclease has the catalytic site for Rec12-oligonucleotide generation and is activated by Ctp1, which plays an additional role in meiotic recombination.

  3. The Third Exon of the Budding Yeast Meiotic Recombination Gene HOP2 Is Required for Calcium-dependent and Recombinase Dmc1-specific Stimulation of Homologous Strand Assimilation*

    PubMed Central

    Chan, Yuen-Ling; Brown, M. Scott; Qin, Daoming; Handa, Naofumi; Bishop, Douglas K.

    2014-01-01

    During meiosis in Saccharomyces cerevisiae, the HOP2 and MND1 genes are essential for recombination. A previous biochemical study has shown that budding yeast Hop2-Mnd1 stimulates the activity of the meiosis-specific strand exchange protein ScDmc1 only 3-fold, whereas analogous studies using mammalian homologs show >30-fold stimulation. The HOP2 gene was recently discovered to contain a second intron that lies near the 3′-end. We show that both HOP2 introns are efficiently spliced during meiosis, forming a predominant transcript that codes for a protein with a C-terminal sequence different from that of the previously studied version of the protein. Using the newly identified HOP2 open reading frame to direct synthesis of wild type Hop2 protein, we show that the Hop2-Mnd1 heterodimer stimulated Dmc1 D-loop activity up to 30-fold, similar to the activity of mammalian Hop2-Mnd1. ScHop2-Mnd1 stimulated ScDmc1 activity in the presence of physiological (micromolar) concentrations of Ca2+ ions, as long as Mg2+ was also present at physiological concentrations, leading us to hypothesize that ScDmc1 protomers bind both cations in the active Dmc1 filament. Co-factor requirements and order-of-addition experiments suggested that Hop2-Mnd1-mediated stimulation of Dmc1 involves a process that follows the formation of functional Dmc1-ssDNA filaments. In dramatic contrast to mammalian orthologs, the stimulatory activity of budding yeast Hop2-Mnd1 appeared to be specific to Dmc1; we observed no Hop2-Mnd1-mediated stimulation of the other budding yeast strand exchange protein Rad51. Together, these results support previous genetic experiments indicating that Hop2-Mnd1 specifically stimulates Dmc1 during meiotic recombination in budding yeast. PMID:24798326

  4. The third exon of the budding yeast meiotic recombination gene HOP2 is required for calcium-dependent and recombinase Dmc1-specific stimulation of homologous strand assimilation.

    PubMed

    Chan, Yuen-Ling; Brown, M Scott; Qin, Daoming; Handa, Naofumi; Bishop, Douglas K

    2014-06-27

    During meiosis in Saccharomyces cerevisiae, the HOP2 and MND1 genes are essential for recombination. A previous biochemical study has shown that budding yeast Hop2-Mnd1 stimulates the activity of the meiosis-specific strand exchange protein ScDmc1 only 3-fold, whereas analogous studies using mammalian homologs show >30-fold stimulation. The HOP2 gene was recently discovered to contain a second intron that lies near the 3'-end. We show that both HOP2 introns are efficiently spliced during meiosis, forming a predominant transcript that codes for a protein with a C-terminal sequence different from that of the previously studied version of the protein. Using the newly identified HOP2 open reading frame to direct synthesis of wild type Hop2 protein, we show that the Hop2-Mnd1 heterodimer stimulated Dmc1 D-loop activity up to 30-fold, similar to the activity of mammalian Hop2-Mnd1. ScHop2-Mnd1 stimulated ScDmc1 activity in the presence of physiological (micromolar) concentrations of Ca(2+) ions, as long as Mg(2+) was also present at physiological concentrations, leading us to hypothesize that ScDmc1 protomers bind both cations in the active Dmc1 filament. Co-factor requirements and order-of-addition experiments suggested that Hop2-Mnd1-mediated stimulation of Dmc1 involves a process that follows the formation of functional Dmc1-ssDNA filaments. In dramatic contrast to mammalian orthologs, the stimulatory activity of budding yeast Hop2-Mnd1 appeared to be specific to Dmc1; we observed no Hop2-Mnd1-mediated stimulation of the other budding yeast strand exchange protein Rad51. Together, these results support previous genetic experiments indicating that Hop2-Mnd1 specifically stimulates Dmc1 during meiotic recombination in budding yeast.

  5. Recombinational landscape of porcine X chromosome and individual variation in female meiotic recombination associated with haplotypes of Chinese pigs

    PubMed Central

    2010-01-01

    Background Variations in recombination fraction (θ) among chromosomal regions, individuals and families have been observed and have an important impact on quantitative trait loci (QTL) mapping studies. Such variations on porcine chromosome X (SSC-X) and on other mammalian chromosome X are rarely explored. The emerging assembly of pig sequence provides exact physical location of many markers, facilitating the study of a fine-scale recombination landscape of the pig genome by comparing a clone-based physical map to a genetic map. Using large offspring of F1 females from two large-scale resource populations (Large White ♂ × Chinese Meishan ♀, and White Duroc ♂ × Chinese Erhualian ♀), we were able to evaluate the heterogeneity in θ for a specific interval among individual F1 females. Results Alignments between the cytogenetic map, radiation hybrid (RH) map, genetic maps and clone map of SSC-X with the physical map of human chromosome X (HSA-X) are presented. The most likely order of 60 markers on SSC-X is inferred. The average recombination rate across SSC-X is of ~1.27 cM/Mb. However, almost no recombination occurred in a large region of ~31 Mb extending from the centromere to Xq21, whereas in the surrounding regions and in the Xq telomeric region a recombination rate of 2.8-3.3 cM/Mb was observed, more than twice the chromosome-wide average rate. Significant differences in θ among F1 females within each population were observed for several chromosomal intervals. The largest variation was observed in both populations in the interval UMNP71-SW1943, or more precisely in the subinterval UMNP891-UMNP93. The individual variation in θ over this subinterval was found associated with F1 females' maternal haplotypes (Chinese pig haplotypes) and independent of paternal haplotype (European pig haplotypes). The θ between UMNP891 and UMNP93 for haplotype 1122 and 4311 differed by more than fourteen-fold (10.3% vs. 0.7%). Conclusions This study reveals marked

  6. Recombination-dependent concatemeric viral DNA replication.

    PubMed

    Lo Piano, Ambra; Martínez-Jiménez, María I; Zecchi, Lisa; Ayora, Silvia

    2011-09-01

    The initiation of viral double stranded (ds) DNA replication involves proteins that recruit and load the replisome at the replication origin (ori). Any block in replication fork progression or a programmed barrier may act as a factor for ori-independent remodelling and assembly of a new replisome at the stalled fork. Then replication initiation becomes dependent on recombination proteins, a process called recombination-dependent replication (RDR). RDR, which is recognized as being important for replication restart and stability in all living organisms, plays an essential role in the replication cycle of many dsDNA viruses. The SPP1 virus, which infects Bacillus subtilis cells, serves as a paradigm to understand the links between replication and recombination in circular dsDNA viruses. SPP1-encoded initiator and replisome assembly proteins control the onset of viral replication and direct the recruitment of host-encoded replisomal components at viral oriL. SPP1 uses replication fork reactivation to switch from ori-dependent θ-type (circle-to-circle) replication to σ-type RDR. Replication fork arrest leads to a double strand break that is processed by viral-encoded factors to generate a D-loop into which a new replisome is assembled, leading to σ-type viral replication. SPP1 RDR proteins are compared with similar proteins encoded by other viruses and their possible in vivo roles are discussed.

  7. Meiotically and mitotically stable inheritance of DNA hypomethylation induced by ddm1 mutation of Arabidopsis thaliana.

    PubMed Central

    Kakutani, T; Munakata, K; Richards, E J; Hirochika, H

    1999-01-01

    In contrast to mammalian epigenetic phenomena, where resetting of gene expression generally occurs in each generation, epigenetic states of plant genes are often stably transmitted through generations. The Arabidopsis mutation ddm1 causes a 70% reduction in genomic 5-methylcytosine level. We have previously shown that the ddm1 mutation results in an accumulation of a variety of developmental abnormalities by slowly inducing heritable changes in other loci. Each of the examined ddm1-induced developmental abnormalities is stably transmitted even when segregated from the potentiating ddm1 mutation. Here, the inheritance of DNA hypomethylation induced by ddm1 was examined in outcross progeny by HPLC and Southern analyses. The results indicate that (i) DDM1 gene function is not necessary during the gametophyte stage, (ii) ddm1 mutation is completely recessive, and (iii) remethylation of sequences hypomethylated by the ddm1 mutation is extremely slow or nonexistent even in wild-type DDM1 backgrounds. The stable transmission of DNA methylation status may be related to the meiotic heritability of the ddm1-induced developmental abnormalities. PMID:9927473

  8. The mouse Spo11 gene is required for meiotic chromosome synapsis.

    PubMed

    Romanienko, P J; Camerini-Otero, R D

    2000-11-01

    The Spo11 protein initiates meiotic recombination by generating DNA double-strand breaks (DSBs) and is required for meiotic synapsis in S. cerevisiae. Surprisingly, Spo11 homologs are dispensable for synapsis in C. elegans and Drosophila yet required for meiotic recombination. Disruption of mouse Spo11 results in infertility. Spermatocytes arrest prior to pachytene with little or no synapsis and undergo apoptosis. We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis. Spo11 localizes to discrete foci during leptotene and to homologously synapsed chromosomes. Other mouse mutants that arrest during meiotic prophase (Atm -/-, Dmc1 -/-, mei1, and Morc(-/-)) showed altered Spo11 protein localization and expression. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis.

  9. Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination

    PubMed Central

    Ziolkowski, Piotr A.; Underwood, Charles J.; Lambing, Christophe; Martinez-Garcia, Marina; Lawrence, Emma J.; Ziolkowska, Liliana; Griffin, Catherine; Choi, Kyuha; Franklin, F. Chris H.; Martienssen, Robert A.; Henderson, Ian R.

    2017-01-01

    During meiosis, homologous chromosomes undergo crossover recombination, which creates genetic diversity and balances homolog segregation. Despite these critical functions, crossover frequency varies extensively within and between species. Although natural crossover recombination modifier loci have been detected in plants, causal genes have remained elusive. Using natural Arabidopsis thaliana accessions, we identified two major recombination quantitative trait loci (rQTLs) that explain 56.9% of crossover variation in Col×Ler F2 populations. We mapped rQTL1 to semidominant polymorphisms in HEI10, which encodes a conserved ubiquitin E3 ligase that regulates crossovers. Null hei10 mutants are haploinsufficient, and, using genome-wide mapping and immunocytology, we show that transformation of additional HEI10 copies is sufficient to more than double euchromatic crossovers. However, heterochromatic centromeres remained recombination-suppressed. The strongest HEI10-mediated crossover increases occur in subtelomeric euchromatin, which is reminiscent of sex differences in Arabidopsis recombination. Our work reveals that HEI10 naturally limits Arabidopsis crossovers and has the potential to influence the response to selection. PMID:28223312

  10. The spatial regulation of meiotic recombination hotspots: are all DSB hotspots crossover hotspots?

    PubMed

    Serrentino, Maria-Elisabetta; Borde, Valérie

    2012-07-15

    A key step for the success of meiosis is programmed homologous recombination, during which crossovers, or exchange of chromosome arms, take place. Crossovers increase genetic diversity but their main function is to ensure accurate chromosome segregation. Defects in crossover number and position produce aneuploidies that represent the main cause of miscarriages and chromosomal abnormalities such as Down's syndrome. Recombination is initiated by the formation of programmed double strand breaks (DSBs), which occur preferentially at places called DSB hotspots. Among all DSBs generated, only a small fraction is repaired by crossover, the other being repaired by other homologous recombination pathways. Crossover maps have been generated in a number of organisms, defining crossover hotspots. With the availability of genome-wide maps of DSBs as well as the ability to measure genetically the repair outcome at several hotspots, it is becoming more and more clear that not all DSB hotspots behave the same for crossover formation, suggesting that chromosomal features distinguish different types of hotspots.

  11. Effects of trans-acting Genetic Modifiers on Meiotic Recombination Across the a1–sh2 Interval of Maize

    PubMed Central

    Yandeau-Nelson, Marna D.; Nikolau, Basil J.; Schnable, Patrick S.

    2006-01-01

    Meiotic recombination rates are potentially affected by cis- and trans-acting factors, i.e., genotype-specific modifiers that do or do not reside in the recombining interval, respectively. Effects of trans modifiers on recombination across the ∼140-kb maize a1–sh2 interval of chromosome 3L were studied in the absence of polymorphic cis factors in three genetically diverse backgrounds into which a sequence-identical a1–sh2 interval had been introgressed. Genetic distances across a1–sh2 varied twofold among genetic backgrounds. Although the existence of regions exhibiting high and low rates of recombination (hot and cold spots, respectively) was conserved across backgrounds, the absolute rates of recombination in these sequence-identical regions differed significantly among backgrounds. In addition, an intergenic hot spot had a higher rate of recombination as compared to the genome average rate of recombination in one background and not in another. Recombination rates across two genetic intervals on chromosome 1 did not exhibit the same relationships among backgrounds as was observed in a1–sh2. This suggests that at least some detected trans-acting factors do not equally affect recombination across the genome. This study establishes that trans modifier(s) polymorphic among genetic backgrounds can increase and decrease recombination in both genic and intergenic regions over relatively small genetic and physical intervals. PMID:16816431

  12. Meiotic Recombination Analyses in Pigs Carrying Different Balanced Structural Chromosomal Rearrangements

    PubMed Central

    Mary, Nicolas; Barasc, Harmonie; Ferchaud, Stéphane; Priet, Aurélia; Calgaro, Anne; Loustau-Dudez, Anne-Marie; Bonnet, Nathalie; Yerle, Martine; Ducos, Alain; Pinton, Alain

    2016-01-01

    Correct pairing, synapsis and recombination between homologous chromosomes are essential for normal meiosis. All these events are strongly regulated, and our knowledge of the mechanisms involved in this regulation is increasing rapidly. Chromosomal rearrangements are known to disturb these processes. In the present paper, synapsis and recombination (number and distribution of MLH1 foci) were studied in three boars (Sus scrofa domestica) carrying different chromosomal rearrangements. One (T34he) was heterozygote for the t(3;4)(p1.3;q1.5) reciprocal translocation, one (T34ho) was homozygote for that translocation, while the third (T34Inv) was heterozygote for both the translocation and a pericentric inversion inv(4)(p1.4;q2.3). All three boars were normal for synapsis and sperm production. This particular situation allowed us to rigorously study the impact of rearrangements on recombination. Overall, the rearrangements induced only minor modifications of the number of MLH1 foci (per spermatocyte or per chromosome) and of the length of synaptonemal complexes for chromosomes 3 and 4. The distribution of MLH1 foci in T34he was comparable to that of the controls. Conversely, the distributions of MLH1 foci on chromosome 4 were strongly modified in boar T34Inv (lack of crossover in the heterosynaptic region of the quadrivalent, and crossover displaced to the chromosome extremities), and also in boar T34ho (two recombination peaks on the q-arms compared with one of higher magnitude in the controls). Analyses of boars T34he and T34Inv showed that the interference was propagated through the breakpoints. A different result was obtained for boar T34ho, in which the breakpoints (transition between SSC3 and SSC4 chromatin on the bivalents) seemed to alter the transmission of the interference signal. Our results suggest that the number of crossovers and crossover interference could be regulated by partially different mechanisms. PMID:27124413

  13. Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation.

    PubMed

    Mahadevaiah, Shantha K; Bourc'his, Déborah; de Rooij, Dirk G; Bestor, Timothy H; Turner, James M A; Burgoyne, Paul S

    2008-07-28

    Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11-null mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

  14. A Link between Meiotic Prophase Progression and CrossoverControl

    SciTech Connect

    Carlton, Peter M.; Farruggio, Alfonso P.; Dernburg, Abby F.

    2005-07-06

    During meiosis, most organisms ensure that homologous chromosomes undergo at least one exchange of DNA, or crossover, to link chromosomes together and accomplish proper segregation. How each chromosome receives a minimum of one crossover is unknown. During early meiosis in Caenorhabditis elegans and many other species, chromosomes adopt a polarized organization within the nucleus, which normally disappears upon completion of homolog synapsis. Mutations that impair synapsis even between a single pair of chromosomes in C. elegans delay this nuclear reorganization. We quantified this delay by developing a classification scheme for discrete stages of meiosis. Immunofluorescence localization of RAD-51 protein revealed that delayed meiotic cells also contained persistent recombination intermediates. Through genetic analysis, we found that this cytological delay in meiotic progression requires double-strand breaks and the function of the crossover-promoting heteroduplex HIM-14 (Msh4) and MSH-5. Failure of X chromosome synapsis also resulted in impaired crossover control on autosomes, which may result from greater numbers and persistence of recombination intermediates in the delayed nuclei. We conclude that maturation of recombination events on chromosomes promotes meiotic progression, and is coupled to the regulation of crossover number and placement. Our results have broad implications for the interpretation of meiotic mutants, as we have shown that asynapsis of a single chromosome pair can exert global effects on meiotic progression and recombination frequency.

  15. Use of a ring chromosome and pulsed-field gels to study interhomolog recombination, double-strand DNA breaks and sister-chromatid exchange in yeast

    SciTech Connect

    Game, J.C. ); Sitney, K.C.; Cook, V.E.; Mortimer, R.K. )

    1989-12-01

    The authors describe a system that uses pulsed-field gels for the physical detection of recombinant DNA molecules, double-strand DNA breaks (DSB) and sister-chromatid exchange in the yeast Saccharomyces cerevisiae. The system makes use of a circular variant of chromosome II (Chr. III). Meiotic recombination between this ring chromosome and a linear homolog produces new molecules of sizes distinguishable on gels from either parental molecule. They demonstrate that these recombinant molecules are not present either in strains with two linear Chr. III molecules or in rad50 mutants, which are defective in meiotic recombination. In conjunction with the molecular endpoints. They present data on the timing of commitment to meiotic recombination scored genetically. They have used x-rays to linearize circular Chr. III, both to develop a sensitive method for measuring frequency of DSB and as a means of detecting double-size circles originating in part from sister-chromatid exchange, which they find to be frequent during meiosis.

  16. Female meiotic sex chromosome inactivation in chicken.

    PubMed

    Schoenmakers, Sam; Wassenaar, Evelyne; Hoogerbrugge, Jos W; Laven, Joop S E; Grootegoed, J Anton; Baarends, Willy M

    2009-05-01

    During meiotic prophase in male mammals, the heterologous X and Y chromosomes remain largely unsynapsed, and meiotic sex chromosome inactivation (MSCI) leads to formation of the transcriptionally silenced XY body. In birds, the heterogametic sex is female, carrying Z and W chromosomes (ZW), whereas males have the homogametic ZZ constitution. During chicken oogenesis, the heterologous ZW pair reaches a state of complete heterologous synapsis, and this might enable maintenance of transcription of Z- and W chromosomal genes during meiotic prophase. Herein, we show that the ZW pair is transiently silenced, from early pachytene to early diplotene using immunocytochemistry and gene expression analyses. We propose that ZW inactivation is most likely achieved via spreading of heterochromatin from the W on the Z chromosome. Also, persistent meiotic DNA double-strand breaks (DSBs) may contribute to silencing of Z. Surprisingly, gammaH2AX, a marker of DSBs, and also the earliest histone modification that is associated with XY body formation in mammalian and marsupial spermatocytes, does not cover the ZW during the synapsed stage. However, when the ZW pair starts to desynapse, a second wave of gammaH2AX accumulates on the unsynapsed regions of Z, which also show a reappearance of the DSB repair protein RAD51. This indicates that repair of meiotic DSBs on the heterologous part of Z is postponed until late pachytene/diplotene, possibly to avoid recombination with regions on the heterologously synapsed W chromosome. Two days after entering diplotene, the Z looses gammaH2AX and shows reactivation. This is the first report of meiotic sex chromosome inactivation in a species with female heterogamety, providing evidence that this mechanism is not specific to spermatogenesis. It also indicates the presence of an evolutionary force that drives meiotic sex chromosome inactivation independent of the final achievement of synapsis.

  17. Single-Stranded DNA Curtains for Studying Homologous Recombination.

    PubMed

    Ma, C J; Steinfeld, J B; Greene, E C

    2017-01-01

    Homologous recombination is an important pathway involved in the repair of double-stranded DNA breaks. Genetic studies form the foundation of our knowledge on homologous recombination. Significant progress has also been made toward understanding the biochemical and biophysical properties of the proteins, complexes, and reaction intermediates involved in this essential DNA repair pathway. However, heterogeneous or transient recombination intermediates remain extremely difficult to assess through traditional ensemble methods, leaving an incomplete mechanistic picture of many steps that take place during homologous recombination. To help overcome some of these limitations, we have established DNA curtain methodologies as an experimental platform for studying homologous DNA recombination in real-time at the single-molecule level. Here, we present a detailed overview describing the preparation and use of single-stranded DNA curtains in applications related to the study of homologous DNA recombination with emphasis on recent work related to the study of the eukaryotic recombinase Rad51.

  18. Meiotic chromosome synapsis in yeast can occur without spo11-induced DNA double-strand breaks.

    PubMed

    Bhuiyan, Hasanuzzaman; Schmekel, Karin

    2004-10-01

    Proper chromosome segregation and formation of viable gametes depend on synapsis and recombination between homologous chromosomes during meiosis. Previous reports have shown that the synaptic structures, the synaptonemal complexes (SCs), do not occur in yeast cells with the SPO11 gene removed. The Spo11 enzyme makes double-strand breaks (DSBs) in the DNA and thereby initiates recombination. The view has thus developed that synapsis in yeast strictly depends on the initiation of recombination. Synapsis in some other species (Drosophila melanogaster and Caenorhabditis elegans) is independent of recombination events, and SCs are found in spo11 mutants. This difference between species led us to reexamine spo11 deletion mutants of yeast. Using antibodies against Zip1, a SC component, we found that a small fraction (1%) of the spo11 null mutant cells can indeed form wild-type-like SCs. We further looked for synapsis in a spo11 mutant strain that accumulates pachytene cells (spo11Delta ndt80Delta), and found that the frequency of cells with apparently complete SC formation was 10%. Other phenotypic criteria, such as spore viability and homologous chromosome juxtaposition measured by FISH labeling of chromosomal markers, agree with several previous reports of the spo11 mutant. Our results demonstrate that although the Spo11-induced DSBs obviously promote synapsis in yeast, the presence of Spo11 is not an absolute requirement for synapsis.

  19. Meiotic process and aneuploidy

    SciTech Connect

    Grell, R.F.

    1985-01-01

    The process of meiosis is analyzed by dissecting it into its component parts using the early oocyte of Drosophila as a model. Entrance of the oocytes into premeiotic interphase signals initiation of DNA replication which continues for 30 h. Coincidentally, extensive synaptonemal complexes appear, averaging 50 ..mu..m (132 h), peaking at 75 ..mu..m (144 h) and continuing into early vitellarial stages. Recombinational response to heat, evidenced by enhancement or induction of exchange, is limited to the S-phase with a peak at 144 h coinciding with maximal extension of the SC. Coincidence of synapsis and recombination response with S at premeiotic interphase is contrary to their conventional localization at meiotic prophase. The interrelationship between exchange and nondisjunction has been clarified by the Distributive Pairing Model of meiosis. Originally revealed through high frequencies of nonrandom assortment of nonhomologous chromosomes, distributive pairing has been shown to follow and to be noncompetitive with exchange, to be based on size-recognition, not homology, and as a raison d'etre, to provide a segregational mechanism for noncrossover homologues. Rearrangements, recombination mutants and aneuploids may contribute noncrossover chromosomes to the distributive pool and so promote the nonhomologous associations responsible for nondisjunction of homologues and regular segregation of nonhomologues. 38 references, 15 figures. (ACR)

  20. Effects of suppressing the DNA mismatch repair system on homeologous recombination in tomato.

    PubMed

    Tam, Sheh May; Hays, John B; Chetelat, Roger T

    2011-12-01

    In plant breeding, the ability to manipulate genetic (meiotic) recombination would be beneficial for facilitating gene transfer from wild relatives of crop plants. The DNA mismatch repair (MMR) system helps maintain genetic integrity by correcting base mismatches that arise via DNA synthesis or damage, and antagonizes recombination between homeologous (divergent) DNA sequences. Previous studies have established that the genomes of cultivated tomato (Solanum lycopersicum) and the wild relative S. lycopersicoides are substantially diverged (homeologous) such that recombination between their chromosomes is strongly reduced. Here, we report the effects on homeologous recombination of suppressing endogenous MMR genes in S. lycopersicum via RNAi-induced silencing of SlMSH2 and SlMSH7 or overexpressing dominant negatives of Arabidopsis MSH2 (AtMSH2-DN) in an alien substitution line (SL-8) of S. lycopersicoides in tomato. We show that certain inhibitions of MMR (RNAi of SlMSH7, AtMSH2-DN) are associated with modest increases in homeologous recombination, ranging from 3.8 to 29.2% (average rate of 17.8%) compared to controls. Unexpectedly, only the AtMSH2-DN proteins but not RNAi-induced silencing of MSH2 was found to increase homeologous recombination. The ratio of single to double crossovers (SCO:DCO ratio) decreased by approximately 50% in progeny of the AtMSH2-DN parents. An increase in the frequency of heterozygous SL-8 plants was also observed in the progeny of the SlMSH7-RNAi parents. Our findings may contribute to acceleration of introgression in cultivated tomato.

  1. Reflections: ethics and the recombinant DNA debate.

    PubMed

    Robb, J W

    1982-01-01

    This essay is an attempt to apply a mode of ethical reflection to questions related to the recombinant DNA debate. The author suggests that adequate ethical analysis must include at least five aspects of a moral situation 1) the nature of the act itself; 2) consequences that result from an action or decision; 3) civil and criminal laws; 4) moral principles; and 5) motivation or intention. This paradigm for analysis is applied to the recombinant DNA scene, particularly as these factors relate to 1) risks vs benefits; 2) laws and regulations; 3) the marketplace; and 4) human genetic intervention. The analysis does not provide answers to these vexing questions, but attempts to raise the consciousness level of the reader concerning significant ethical issues. Undergirding the discussion is the humanistic theme of the primacy of the concern for the public good, and that freedom of inquiry can only survive in a socially conscious context. Fear of the unknown is no reason for prohibiting DNA research, but since the stakes are high as new developments emerge that pertain to the genetic restructuring of the human species, the author suggests that caution and responsible judgment are mandatory. Since ethics is concerned with ideal moral judgments, and applied ethics relates to the real world in which we function, the demand for clear and careful thought concerning the short-term and long-term consequences of our work is a primary ingredient of what it means to be responsible. In the final analysis, ethical responsibility rests on the individual; without a sense of personal integrity and what that implies about oneself and society, blatant opportunism can easily become the generally accepted mode of behavior and decision. If this occurs, traditional ethical concerns become irrelevant.

  2. Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerevisiae.

    PubMed Central

    Wagstaff, J E; Klapholz, S; Waddell, C S; Jensen, L; Esposito, R E

    1985-01-01

    We used haploid yeast cells that express both the MATa and MAT alpha mating-type alleles and contain the spo13-1 mutation to characterize meiotic recombination within single, unpaired chromosomes in Rec+ and Rec- Saccharomyces cerevisiae. In Rec+ haploids, as in diploids, intrachromosomal recombination in the ribosomal DNA was detected in 2 to 6% of meiotic divisions, and most events were unequal reciprocal sister chromatid exchange (SCE). By contrast, intrachromosomal recombination between duplicated copies of the his4 locus occurred in approximately 30% of haploid meiotic divisions, a frequency much higher than that reported in diploids; only about one-half of the events were unequal reciprocal SCE. The spo11-1 mutation, which virtually eliminates meiotic exchange between homologs in diploid meiosis, reduced the frequency of intrachromosomal recombination in both the ribosomal DNA and the his4 duplication during meiosis by 10- to greater than 50-fold. This Rec- mutation affected all forms of recombination within chromosomes: unequal reciprocal SCE, reciprocal intrachromatid exchange, and gene conversion. Intrachromosomal recombination in spo11-1 haploids was restored by transformation with a plasmid containing the wild-type SPO11 gene. Mitotic intrachromosomal recombination frequencies were unaffected by spo11-1. This is the first demonstration of a gene product required for recombination between homologs as well as recombination within chromosomes during meiosis. Images PMID:3915779

  3. [Analysis of the meiotic recombination frequency in transgenic tomato hybrids expressing recA and NLS-recA-licBM3 genes].

    PubMed

    Komakhin, R A; Komakhina, V V; Miliukova, N A; Zhuchenko, A A

    2012-01-01

    To study and induce meiotic recombination in plants, we generated and analyzed transgenic tomato hybrids F1-RecA and F1-NLS-recA-LicBM3 expressing, respectively, the recA gene of Escherichia coli and the NLS-recA-licBM3 gene. It was found that the recA and NLS-recA-licBM3 genes are inherited through the maternal and paternal lineages, they have no selective influence on the pollen and are contained in tomato F1-RecA and F1-NLS-RecA-LicBM3 hybrids outside the second chromosome in the hemizygous state. The comparative analysis of the meiotic recombination frequency (rf) in the progenies of the transgenic and nontransgenic hybrids showed that only the expression of the recA gene of E. coli in cells of the F1-RecA plants produced a 1.2-1.5-fold increase in the frequency of recombination between some linked marker genes of the second chromosome of tomato.

  4. High-Resolution Global Analysis of the Influences of Bas1 and Ino4 Transcription Factors on Meiotic DNA Break Distributions in Saccharomyces cerevisiae.

    PubMed

    Zhu, Xuan; Keeney, Scott

    2015-10-01

    Meiotic recombination initiates with DNA double-strand breaks (DSBs) made by Spo11. In Saccharomyces cerevisiae, many DSBs occur in "hotspots" coinciding with nucleosome-depleted gene promoters. Transcription factors (TFs) stimulate DSB formation in some hotspots, but TF roles are complex and variable between locations. Until now, available data for TF effects on global DSB patterns were of low spatial resolution and confined to a single TF. Here, we examine at high resolution the contributions of two TFs to genome-wide DSB distributions: Bas1, which was known to regulate DSB activity at some loci, and Ino4, for which some binding sites were known to be within strong DSB hotspots. We examined fine-scale DSB distributions in TF mutant strains by deep sequencing oligonucleotides that remain covalently bound to Spo11 as a byproduct of DSB formation, mapped Bas1 and Ino4 binding sites in meiotic cells, evaluated chromatin structure around DSB hotspots, and measured changes in global messenger RNA levels. Our findings show that binding of these TFs has essentially no predictive power for DSB hotspot activity and definitively support the hypothesis that TF control of DSB numbers is context dependent and frequently indirect. TFs often affected the fine-scale distributions of DSBs within hotspots, and when seen, these effects paralleled effects on local chromatin structure. In contrast, changes in DSB frequencies in hotspots did not correlate with quantitative measures of chromatin accessibility, histone H3 lysine 4 trimethylation, or transcript levels. We also ruled out hotspot competition as a major source of indirect TF effects on DSB distributions. Thus, counter to prevailing models, roles of these TFs on DSB hotspot strength cannot be simply explained via chromatin "openness," histone modification, or compensatory interactions between adjacent hotspots.

  5. A Quality Control Mechanism Coordinates Meiotic Prophase Events to Promote Crossover Assurance

    PubMed Central

    Deshong, Alison J.; Ye, Alice L.; Lamelza, Piero; Bhalla, Needhi

    2014-01-01

    Meiotic chromosome segregation relies on homologous chromosomes being linked by at least one crossover, the obligate crossover. Homolog pairing, synapsis and meiosis specific DNA repair mechanisms are required for crossovers but how they are coordinated to promote the obligate crossover is not well understood. PCH-2 is a highly conserved meiotic AAA+-ATPase that has been assigned a variety of functions; whether these functions reflect its conserved role has been difficult to determine. We show that PCH-2 restrains pairing, synapsis and recombination in C. elegans. Loss of pch-2 results in the acceleration of synapsis and homolog-dependent meiotic DNA repair, producing a subtle increase in meiotic defects, and suppresses pairing, synapsis and recombination defects in some mutant backgrounds. Some defects in pch-2 mutants can be suppressed by incubation at lower temperature and these defects increase in frequency in wildtype worms grown at higher temperature, suggesting that PCH-2 introduces a kinetic barrier to the formation of intermediates that support pairing, synapsis or crossover recombination. We hypothesize that this kinetic barrier contributes to quality control during meiotic prophase. Consistent with this possibility, defects in pch-2 mutants become more severe when another quality control mechanism, germline apoptosis, is abrogated or meiotic DNA repair is mildly disrupted. PCH-2 is expressed in germline nuclei immediately preceding the onset of stable homolog pairing and synapsis. Once chromosomes are synapsed, PCH-2 localizes to the SC and is removed in late pachytene, prior to SC disassembly, correlating with when homolog-dependent DNA repair mechanisms predominate in the germline. Indeed, loss of pch-2 results in premature loss of homolog access. Altogether, our data indicate that PCH-2 coordinates pairing, synapsis and recombination to promote crossover assurance. Specifically, we propose that the conserved function of PCH-2 is to destabilize pairing

  6. Spatiotemporal Asymmetry of the Meiotic Program Underlies the Predominantly Distal Distribution of Meiotic Crossovers in Barley[W

    PubMed Central

    Higgins, James D.; Perry, Ruth M.; Barakate, Abdellah; Ramsay, Luke; Waugh, Robbie; Halpin, Claire; Armstrong, Susan J.; Franklin, F. Chris H.

    2012-01-01

    Meiosis involves reciprocal exchange of genetic information between homologous chromosomes to generate new allelic combinations. In cereals, the distribution of genetic crossovers, cytologically visible as chiasmata, is skewed toward the distal regions of the chromosomes. However, many genes are known to lie within interstitial/proximal regions of low recombination, creating a limitation for breeders. We investigated the factors underlying the pattern of chiasma formation in barley (Hordeum vulgare) and show that chiasma distribution reflects polarization in the spatiotemporal initiation of recombination, chromosome pairing, and synapsis. Consequently, meiotic progression in distal chromosomal regions occurs in coordination with the chromatin cycles that are a conserved feature of the meiotic program. Recombination initiation in interstitial and proximal regions occurs later than distal events, is not coordinated with the cycles, and rarely progresses to form chiasmata. Early recombination initiation is spatially associated with early replicating, euchromatic DNA, which is predominately found in distal regions. We demonstrate that a modest temperature shift is sufficient to alter meiotic progression in relation to the chromosome cycles. The polarization of the meiotic processes is reduced and is accompanied by a shift in chiasma distribution with an increase in interstitial and proximal chiasmata, suggesting a potential route to modify recombination in cereals. PMID:23104831

  7. Recombination at the DNA level. Abstracts

    SciTech Connect

    Not Available

    1984-01-01

    Abstracts of papers in the following areas are presented: (1) chromosome mechanics; (2) yeast systems; (3) mammalian homologous recombination; (4) transposons; (5) Mu; (6) plant transposons/T4 recombination; (7) topoisomerase, resolvase, and gyrase; (8) Escherichia coli general recombination; (9) recA; (10) repair; (11) eucaryotic enzymes; (12) integration and excision of bacteriophage; (13) site-specific recombination; and (14) recombination in vitro. (ACR)

  8. Antiviral protein Ski8 is a direct partner of Spo11 in meiotic DNA break formation, independent of its cytoplasmic role in RNA metabolism.

    PubMed

    Arora, Charanjit; Kee, Kehkooi; Maleki, Shohreh; Keeney, Scott

    2004-02-27

    Meiotic recombination initiates with double-strand breaks (DSBs) catalyzed by Spo11 in conjunction with accessory proteins whose roles are not understood. Two-hybrid analysis reveals a network of interactions connecting the yeast DSB proteins to one another. Of these proteins, Ski8 was known to function in cytoplasmic RNA metabolism, suggesting that its role in recombination might be indirect. However, obligate partners of Ski8 in RNA metabolism are dispensable for recombination and Ski8 relocalizes to the nucleus and associates with chromosomes specifically during meiosis. Interaction of Ski8 with Spo11 is essential for DSB formation and Ski8 relocalization. Thus, Ski8 plays distinct roles in RNA metabolism and, as a direct partner of Spo11, in DSB formation. Ski8 works with Spo11 to recruit other DSB proteins to meiotic chromosomes, implicating Ski8 as a scaffold protein mediating assembly of a multiprotein complex essential for DSB formation.

  9. 76 FR 3150 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-19

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines...: The NIH Guidelines currently require that recombinant DNA experiments designed to create...

  10. 75 FR 42114 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-20

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH... transgenic rodents by recombinant DNA technology must be registered with the Institutional...

  11. Prdm9, a major determinant of meiotic recombination hotspots, is not functional in dogs and their wild relatives, wolves and coyotes.

    PubMed

    Muñoz-Fuentes, Violeta; Di Rienzo, Anna; Vilà, Carles

    2011-01-01

    Meiotic recombination is a fundamental process needed for the correct segregation of chromosomes during meiosis in sexually reproducing organisms. In humans, 80% of crossovers are estimated to occur at specific areas of the genome called recombination hotspots. Recently, a protein called PRDM9 was identified as a major player in determining the location of genome-wide meiotic recombination hotspots in humans and mice. The origin of this protein seems to be ancient in evolutionary time, as reflected by its fairly conserved structure in lineages that diverged over 700 million years ago. Despite its important role, there are many animal groups in which Prdm9 is absent (e.g. birds, reptiles, amphibians, diptera) and it has been suggested to have disruptive mutations and thus to be a pseudogene in dogs. Because of the dog's history through domestication and artificial selection, we wanted to confirm the presence of a disrupted Prdm9 gene in dogs and determine whether this was exclusive of this species or whether it also occurred in its wild ancestor, the wolf, and in a close relative, the coyote. We sequenced the region in the dog genome that aligned to the last exon of the human Prdm9, containing the entire zinc finger domain, in 4 dogs, 17 wolves and 2 coyotes. Our results show that the three canid species possess mutations that likely make this gene non functional. Because these mutations are shared across the three species, they must have appeared prior to the split of the wolf and the coyote, millions of years ago, and are not related to domestication. In addition, our results suggest that in these three canid species recombination does not occur at hotspots or hotspot location is controlled through a mechanism yet to be determined.

  12. Double-strand break repair on sex chromosomes: challenges during male meiotic prophase

    PubMed Central

    Lu, Lin-Yu; Yu, Xiaochun

    2015-01-01

    During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with many DNA damage response proteins and form a unique chromatin structure known as the XY body. Interestingly, associated with the prolonged DSB repair, transcription is repressed in the XY body but not in autosomes, a phenomenon known as meiotic sex chromosome inactivation (MSCI), which is critical for male meiosis. Here using mice as model organisms, we briefly summarize recent progress on DSB repair in meiotic prophase and focus on the mechanism and function of DNA damage response in the XY body. PMID:25565522

  13. Double-strand break repair on sex chromosomes: challenges during male meiotic prophase.

    PubMed

    Lu, Lin-Yu; Yu, Xiaochun

    2015-01-01

    During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with many DNA damage response proteins and form a unique chromatin structure known as the XY body. Interestingly, associated with the prolonged DSB repair, transcription is repressed in the XY body but not in autosomes, a phenomenon known as meiotic sex chromosome inactivation (MSCI), which is critical for male meiosis. Here using mice as model organisms, we briefly summarize recent progress on DSB repair in meiotic prophase and focus on the mechanism and function of DNA damage response in the XY body.

  14. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA.

    PubMed Central

    Young, R A; Bloom, B R; Grosskinsky, C M; Ivanyi, J; Thomas, D; Davis, R W

    1985-01-01

    A recombinant DNA strategy has been used systematically to survey the Mycobacterium tuberculosis genome for sequences that encode specific antigens detected by monoclonal antibodies. M. tuberculosis genomic DNA fragments with randomly generated endpoints were used to construct a large lambda gt11 recombinant DNA expression library. Sufficient numbers of recombinants were produced to contain inserts whose endpoints occur at nearly every base pair in the pathogen genome. Protein antigens specified by linear segments of pathogen DNA and produced by the recombinant phage of Escherichia coli were screened with monoclonal antibody probes. This approach was coupled with an improved detection method for gene isolation using antibodies to clonally isolate DNA sequences that specify polypeptide components of M. tuberculosis. The methodology described here, which is applicable to other pathogens, offers possibilities for the development of more sensitive and specific immunodiagnostic and seroepidemiological tests for tuberculosis and, ultimately, for the development of more effective vaccines. Images PMID:2581251

  15. Regulation of DNA strand exchange in homologous recombination.

    PubMed

    Holthausen, J Thomas; Wyman, Claire; Kanaar, Roland

    2010-12-10

    Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate.

  16. Transcript-RNA-templated DNA recombination and repair.

    PubMed

    Keskin, Havva; Shen, Ying; Huang, Fei; Patel, Mikir; Yang, Taehwan; Ashley, Katie; Mazin, Alexander V; Storici, Francesca

    2014-11-20

    Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA-DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity.

  17. Recent advances in yeast molecular biology: recombinant DNA. [Lead abstract

    SciTech Connect

    Not Available

    1982-09-01

    Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis. (KRM)

  18. Meiotic DNA double-strand breaks and chromosome asynapsis in mice are monitored by distinct HORMAD2-independent and -dependent mechanisms.

    PubMed

    Wojtasz, Lukasz; Cloutier, Jeffrey M; Baumann, Marek; Daniel, Katrin; Varga, János; Fu, Jun; Anastassiadis, Konstantinos; Stewart, A Francis; Reményi, Attila; Turner, James M A; Tóth, Attila

    2012-05-01

    Meiotic crossover formation involves the repair of programmed DNA double-strand breaks (DSBs) and synaptonemal complex (SC) formation. Completion of these processes must precede the meiotic divisions in order to avoid chromosome abnormalities in gametes. Enduring key questions in meiosis have been how meiotic progression and crossover formation are coordinated, whether inappropriate asynapsis is monitored, and whether asynapsis elicits prophase arrest via mechanisms that are distinct from the surveillance of unrepaired DNA DSBs. We disrupted the meiosis-specific mouse HORMAD2 (Hop1, Rev7, and Mad2 domain 2) protein, which preferentially associates with unsynapsed chromosome axes. We show that HORMAD2 is required for the accumulation of the checkpoint kinase ATR along unsynapsed axes, but not at DNA DSBs or on DNA DSB-associated chromatin loops. Consistent with the hypothesis that ATR activity on chromatin plays important roles in the quality control of meiotic prophase, HORMAD2 is required for the elimination of the asynaptic Spo11(-/-), but not the asynaptic and DSB repair-defective Dmc1(-/-) oocytes. Our observations strongly suggest that HORMAD2-dependent recruitment of ATR to unsynapsed chromosome axes constitutes a mechanism for the surveillance of asynapsis. Thus, we provide convincing evidence for the existence of a distinct asynapsis surveillance mechanism that safeguards the ploidy of the mammalian germline.

  19. Recombinant DNA production of spider silk proteins

    PubMed Central

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-01-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

  20. Recombinant DNA production of spider silk proteins.

    PubMed

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-11-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.

  1. Roles of DNA helicases in the mediation and regulation of homologous recombination.

    PubMed

    Daley, James M; Niu, Hengyao; Sung, Patrick

    2013-01-01

    Homologous recombination (HR) is an evolutionarily conserved process that eliminates DNA double-strand breaks from chromosomes, repairs injured DNA replication forks, and helps orchestrate meiotic chromosome segregation. Recent studies have shown that DNA helicases play multifaceted roles in HR mediation and regulation. In particular, the S. cerevisiae Sgs1 helicase and its human ortholog BLM helicase are involved in not only the resection of the primary lesion to generate single-stranded DNA to prompt the assembly of the HR machinery, but they also function in somatic cells to suppress the formation of chromosome arm crossovers during HR. On the other hand, the S. cerevisiae Mph1 and Srs2 helicases, and their respective functional equivalents in other eukaryotes, suppress spurious HR events and favor the formation of noncrossovers via distinct mechanisms. Thus, the functional integrity of the HR process and HR outcomes are dependent upon these helicase enzymes. Since mutations in some of these helicases lead to cancer predisposition in humans and mice, studies on them have clear relevance to human health and disease.

  2. Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

    PubMed

    Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

    2015-01-01

    Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries.

  3. Nuclear Localization of PRDM9 and Its Role in Meiotic Chromatin Modifications and Homologous Synapsis

    PubMed Central

    Sun, Fengyun; Fujiwara, Yasuhiro; Reinholdt, Laura G.; Hu, Jianjun; Saxl, Ruth L.; Baker, Christopher L.; Petkov, Petko M.; Paigen, Kenneth; Handel, Mary Ann

    2015-01-01

    Developmental progress of germ cells through meiotic phases is closely tied to ongoing meiotic recombination. In mammals, recombination preferentially occurs in genomic regions known as hotspots; the protein that activates these hotspots is PRDM9, containing a genetically variable zinc-finger domain and a PR-SET domain with histone H3K4 trimethyltransferase activity. PRDM9 is required for fertility in mice, but little is known about its localization and developmental dynamics. Application of spermatogenic stage-specific markers demonstrates that PRDM9 accumulates in male germ-cell nuclei at pre-leptonema to early leptonema, but is no longer detectable in nuclei by late zygonema. By the pachytene stage, PRDM9-dependent histone H3K4 trimethyl marks on hotspots also disappear. PRDM9 localizes to nuclei concurrently with the deposition of meiotic cohesin complexes, but is not required for incorporation of cohesin complex proteins into chromosomal axial elements, or accumulation of normal numbers of RAD51 foci on meiotic chromatin by late zygonema. Germ cells lacking PRDM9 exhibit inefficient homology recognition and synapsis, with aberrant repair of meiotic DNA double-strand breaks and transcriptional abnormalities characteristic of meiotic silencing of unsynapsed chromatin. Together, these results on the developmental time course for nuclear localization of PRDM9 establish its direct window of function, and demonstrate the independence of chromosome axial element formation from the concurrent PRDM9-mediated activation of recombination hotspots. PMID:25894966

  4. Nuclear localization of PRDM9 and its role in meiotic chromatin modifications and homologous synapsis.

    PubMed

    Sun, Fengyun; Fujiwara, Yasuhiro; Reinholdt, Laura G; Hu, Jianjun; Saxl, Ruth L; Baker, Christopher L; Petkov, Petko M; Paigen, Kenneth; Handel, Mary Ann

    2015-09-01

    Developmental progress of germ cells through meiotic phases is closely tied to ongoing meiotic recombination. In mammals, recombination preferentially occurs in genomic regions known as hotspots; the protein that activates these hotspots is PRDM9, containing a genetically variable zinc finger (ZNF) domain and a PR-SET domain with histone H3K4 trimethyltransferase activity. PRDM9 is required for fertility in mice, but little is known about its localization and developmental dynamics. Application of spermatogenic stage-specific markers demonstrates that PRDM9 accumulates in male germ cell nuclei at pre-leptonema to early leptonema but is no longer detectable in nuclei by late zygonema. By the pachytene stage, PRDM9-dependent histone H3K4 trimethyl marks on hotspots also disappear. PRDM9 localizes to nuclei concurrently with the deposition of meiotic cohesin complexes, but is not required for incorporation of cohesin complex proteins into chromosomal axial elements, or accumulation of normal numbers of RAD51 foci on meiotic chromatin by late zygonema. Germ cells lacking PRDM9 exhibit inefficient homology recognition and synapsis, with aberrant repair of meiotic DNA double-strand breaks and transcriptional abnormalities characteristic of meiotic silencing of unsynapsed chromatin. Together, these results on the developmental time course for nuclear localization of PRDM9 establish its direct window of function and demonstrate the independence of chromosome axial element formation from the concurrent PRDM9-mediated activation of recombination hotspots.

  5. Most meiotic CAG repeat tract-length alterations in yeast are SPO11 dependent.

    PubMed

    Jankowski, C; Nag, Dilip K

    2002-03-01

    The expansion of trinucleotide repeat sequences associated with hereditary neurological diseases is believed from earlier studies to be due to errors in DNA replication. However, more recent studies have indicated that recombination may play a significant role in triplet repeat expansion. CAG repeat tracts have been shown to induce double-strand breaks (DSBs) during meiosis in yeast, and DSB formation is dependent on the meiotic recombination machinery. The rate of meiotic instability is several fold higher than mitotic instability. To determine whether DSB repair is responsible for the high rate of repeat tract-length alterations, the frequencies of meiotic repeat-tract instability were compared in wild-type and spo11 mutant strains. In the spo11 background, the rate of meiotic repeat-tract instability remained at the mitotic level, suggesting that meiotic alterations of CAG repeat tracts in yeast occur by the recombination mechanism. Several of these meiotic tract-length alterations are due to DSB repair involving use of the sister chromatid as a template.

  6. Efficient detection of unpaired DNA requires a member of the rad54-like family of homologous recombination proteins.

    PubMed

    Samarajeewa, Dilini A; Sauls, Pegan A; Sharp, Kevin J; Smith, Zachary J; Xiao, Hua; Groskreutz, Katie M; Malone, Tyler L; Boone, Erin C; Edwards, Kevin A; Shiu, Patrick K T; Larson, Erik D; Hammond, Thomas M

    2014-11-01

    Meiotic silencing by unpaired DNA (MSUD) is a process that detects unpaired regions between homologous chromosomes and silences them for the duration of sexual development. While the phenomenon of MSUD is well recognized, the process that detects unpaired DNA is poorly understood. In this report, we provide two lines of evidence linking unpaired DNA detection to a physical search for DNA homology. First, we have found that a putative SNF2-family protein (SAD-6) is required for efficient MSUD in Neurospora crassa. SAD-6 is closely related to Rad54, a protein known to facilitate key steps in the repair of double-strand breaks by homologous recombination. Second, we have successfully masked unpaired DNA by placing identical transgenes at slightly different locations on homologous chromosomes. This masking falls apart when the distance between the transgenes is increased. We propose a model where unpaired DNA detection during MSUD is achieved through a spatially constrained search for DNA homology. The identity of SAD-6 as a Rad54 paralog suggests that this process may be similar to the searching mechanism used during homologous recombination.

  7. Replication and recombination factors contributing to recombination-dependent bypass of DNA lesions by template switch.

    PubMed

    Vanoli, Fabio; Fumasoni, Marco; Szakal, Barnabas; Maloisel, Laurent; Branzei, Dana

    2010-11-11

    Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to

  8. Rogue athletes and recombinant DNA technology: challenges for doping control.

    PubMed

    Azzazy, Hassan M E; Mansour, Mai M H

    2007-10-01

    The quest for athletic excellence holds no limit for some athletes, and the advances in recombinant DNA technology have handed these athletes the ultimate doping weapons: recombinant proteins and gene doping. Some detection methods are now available for several recombinant proteins that are commercially available as pharmaceuticals and being abused by dopers. However, researchers are struggling to come up with efficient detection methods in preparation for the imminent threat of gene doping, expected in the 2008 Olympics. This Forum article presents the main detection strategies for recombinant proteins and the forthcoming detection strategies for gene doping as well as the prime analytical challenges facing them.

  9. Pair-wise linkage disequilibrium decay among linked loci suggests meiotic recombination in natural populations of Sclerotinia sclerotiorum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Both clonal and recombining population structures have been reported in Sclerotinia sclerotiorum populations around the world. Association of independent and putatively unlinked markers indicates clonal population structure, whereas random association of the markers suggests recombination and outcro...

  10. Evidence for Recombination of Mitochondrial DNA in Triploid Crucian Carp

    PubMed Central

    Guo, Xinhong; Liu, Shaojun; Liu, Yun

    2006-01-01

    In this study, we report the complete mitochondrial DNA (mtDNA) sequences of the allotetraploid and triploid crucian carp and compare the complete mtDNA sequences between the triploid crucian carp and its female parent Japanese crucian carp and between the triploid crucian carp and its male parent allotetraploid. Our results indicate that the complete mtDNA nucleotide identity (98%) between the triploid crucian carp and its male parent allotetraploid was higher than that (93%) between the triploid crucian carp and its female parent Japanese crucian carp. Moreover, the presence of a pattern of identity and difference at synonymous sites of mitochondrial genomes between the triploid crucian carp and its parents provides direct evidence that triploid crucian carp possessed the recombination mtDNA fragment (12,759 bp) derived from the paternal fish. These results suggest that mtDNA recombination was derived from the fusion of the maternal and paternal mtDNAs. Compared with the haploid egg with one set of genome from the Japanese crucian carp, the diploid sperm with two sets of genomes from the allotetraploid could more easily make its mtDNA fuse with the mtDNA of the haploid egg. In addition, the triple hybrid nature of the triploid crucian carp probably allowed its better mtDNA recombination. In summary, our results provide the first evidence of mtDNA combination in polyploid fish. PMID:16322508

  11. Purification, folding, and characterization of Rec12 (Spo11) meiotic recombinase of fission yeast.

    PubMed

    Wu, Heng; Gao, Jun; Sharif, Wallace D; Davidson, Mari K; Wahls, Wayne P

    2004-11-01

    Meiotic recombination is initiated by controlled dsDNA breaks (DSBs). Rec12 (Spo11) protein of fission yeast is essential for the formation of meiotic DSBs in vivo, for meiotic recombination, and for segregation of chromosomes during meiosis I. Rec12 is orthologous to Top6A topoisomerase of Archaea and is likely the catalytic subunit of a meiotic recombinase that introduces recombinogenic DSBs. However, despite intensive effort, it has not been possible to produce Rec12 protein in a soluble form required to permit biochemical analyses of function. To obtain purified Rec12 protein for in vitro studies, a rec12(+) cDNA was generated, cloned into vector pET15b(+), and expressed in Escherichia coli. Rec12 protein was produced at moderate levels and it partitioned into insoluble fractions of whole-cell extracts. The protein was enriched based upon its differential solubility in two different denaturants and was further purified by column chromatography. A combinatorial, fractional, factorial approach was used to identify conditions under which Rec12 protein could be refolded. Four parameters were most important and, following optimization, soluble Rec12 protein was obtained. Gel filtration demonstrated that refolded Rec12 protein exists as a monomer in solution, suggesting that additional proteins may be required to assemble biologically-active Rec12 dimers, as inferred previously from genetic data [Cell Chromosome 1 (2002) 1]. The production of refolded Rec12 in a soluble form will allow for characterization in vitro of this key meiotic recombination enzyme.

  12. A mutation in the FHA domain of Coprinus cinereus Nbs1 Leads to Spo11-independent meiotic recombination and chromosome segregation.

    PubMed

    Crown, K Nicole; Savytskyy, Oleksandr P; Malik, Shehre-Banoo; Logsdon, John; Williams, R Scott; Tainer, John A; Zolan, Miriam E

    2013-11-06

    Nbs1, a core component of the Mre11-Rad50-Nbs1 complex, plays an essential role in the cellular response to DNA double-strand breaks (DSBs) and poorly understood roles in meiosis. We used the basidiomycete Coprinus cinereus to examine the meiotic roles of Nbs1. We identified the C. cinereus nbs1 gene and demonstrated that it corresponds to a complementation group previously known as rad3. One allele, nbs1-2, harbors a point mutation in the Nbs1 FHA domain and has a mild spore viability defect, increased frequency of meiosis I nondisjunction, and an altered crossover distribution. The nbs1-2 strain enters meiosis with increased levels of phosphorylated H2AX, which we hypothesize represent unrepaired DSBs formed during premeiotic replication. In nbs1-2, there is no apparent induction of Spo11-dependent DSBs during prophase. We propose that replication-dependent DSBs, resulting from defective replication fork protection and processing by the Mre11-Rad50-Nbs1 complex, are competent to form meiotic crossovers in C. cinereus, and that these crossovers lead to high levels of faithful chromosome segregation. In addition, although crossover distribution is altered in nbs1-2, the majority of crossovers were found in subtelomeric regions, as in wild-type. Therefore, the location of crossovers in C. cinereus is maintained when DSBs are induced via a Spo11-independent mechanism.

  13. Retroviral Integrase Structure and DNA Recombination Mechanism

    PubMed Central

    Engelman, Alan; Cherepanov, Peter

    2015-01-01

    SUMMARY Due to the importance of human immunodeficiency virus type 1 (HIV-1) integrase as a drug target, the biochemistry and structural aspects of retroviral DNA integration have been the focus of intensive research during the past three decades. The retroviral integrase enzyme acts on the linear double-stranded viral DNA product of reverse transcription. Integrase cleaves specific phosphodiester bonds near the viral DNA ends during the 3′ processing reaction. The enzyme then uses the resulting viral DNA 3′-OH groups during strand transfer to cut chromosomal target DNA, which simultaneously joins both viral DNA ends to target DNA 5′-phosphates. Both reactions proceed via direct transesterification of scissile phosphodiester bonds by attacking nucleophiles: a water molecule for 3′ processing, and the viral DNA 3′-OH for strand transfer. X-ray crystal structures of prototype foamy virus integrase-DNA complexes revealed the architectures of the key nucleoprotein complexes that form sequentially during the integration process and explained the roles of active site metal ions in catalysis. X-ray crystallography furthermore elucidated the mechanism of action of HIV-1 integrase strand transfer inhibitors, which are currently used to treat AIDS patients, and provided valuable insights into the mechanisms of viral drug resistance. PMID:25705574

  14. Absence of SUN-domain protein Slp1 blocks karyogamy and switches meiotic recombination and synapsis from homologs to sister chromatids

    PubMed Central

    Vasnier, Christelle; de Muyt, Arnaud; Zhang, Liangran; Tessé, Sophie; Kleckner, Nancy E.; Zickler, Denise; Espagne, Eric

    2014-01-01

    Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid–Sad1p, UNC-84–domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid “twin” nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly. PMID:25210014

  15. Absence of SUN-domain protein Slp1 blocks karyogamy and switches meiotic recombination and synapsis from homologs to sister chromatids.

    PubMed

    Vasnier, Christelle; de Muyt, Arnaud; Zhang, Liangran; Tessé, Sophie; Kleckner, Nancy E; Zickler, Denise; Espagne, Eric

    2014-09-23

    Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid-Sad1p, UNC-84-domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid "twin" nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly.

  16. Meiotic silencing and fragmentation of the male germline restricted chromosome in zebra finch.

    PubMed

    Schoenmakers, Sam; Wassenaar, Evelyne; Laven, Joop S E; Grootegoed, J Anton; Baarends, Willy M

    2010-06-01

    During male meiotic prophase in mammals, X and Y are in a largely unsynapsed configuration, which is thought to trigger meiotic sex chromosome inactivation (MSCI). In avian species, females are ZW, and males ZZ. Although Z and W in chicken oocytes show complete, largely heterologous synapsis, they too undergo MSCI, albeit only transiently. The W chromosome is already inactive in early meiotic prophase, and inactive chromatin marks may spread on to the Z upon synapsis. Mammalian MSCI is considered as a specialised form of the general meiotic silencing mechanism, named meiotic silencing of unsynapsed chromatin (MSUC). Herein, we studied the avian form of MSUC, by analysing the behaviour of the peculiar germline restricted chromosome (GRC) that is present as a single copy in zebra finch spermatocytes. In the female germline, this chromosome is present in two copies, which normally synapse and recombine. In contrast, during male meiosis, the single GRC is always eliminated. We found that the GRC in the male germline is silenced from early leptotene onwards, similar to the W chromosome in avian oocytes. The GRC remains largely unsynapsed throughout meiotic prophase I, although patches of SYCP1 staining indicate that part of the GRC may self-synapse. In addition, the GRC is largely devoid of meiotic double strand breaks. We observed a lack of the inner centromere protein INCENP on the GRC and elimination of the GRC following metaphase I. Subsequently, the GRC forms a micronucleus in which the DNA is fragmented. We conclude that in contrast to MSUC in mammals, meiotic silencing of this single chromosome in the avian germline occurs prior to, and independent of DNA double strand breaks and chromosome pairing, hence we have named this phenomenon meiotic silencing prior to synapsis (MSPS).

  17. Analysis of four microsatellite markers on the long arm of chromosome 9 by meiotic recombination in flow-sorted single sperm

    SciTech Connect

    Furlong, R.A.; Goudie, D.R.; Carter, N.P.; Lyall, J.E.W.; Affara, N.A.; Ferguson-Smith, M.A. )

    1993-06-01

    Meiotic recombination in flow-sorted single sperm was used to analyze four highly polymorphic microsatellite markers on the long arm of chromosome 9. The microsatellites comprised three tightly linked markers: 9CMP1 (D9S109), 9CMP2 (D9S127), and D9S53, which map to 9q31, and a reference marker, ASS, which is located in 9q34.1. Haplotypes of single sperm were assessed by using PCR in a single-step multiplex reaction to amplify each locus. Recombinant haplotypes were identified by their relative infrequency and were analyzed using THREELOC, a maximum-likelihood-analysis program, and an adaptation of CRI-MAP. The most likely order of these markers was cen-D9S109-D9S127-D9S53-ASS-tel with D9S109, D9S127, and D9S53 being separated by a genetic distance of approximately 3%. The order of the latter three markers did not however achieve statistical significance using the THREELOC program. 21 refs., 2 figs., 4 tabs.

  18. Historical perspectives pertaining to the NIH Recombinant DNA Advisory Committee.

    PubMed

    Wivel, Nelson A

    2014-01-01

    Science is host to a constantly emerging series of new paradigms, and it is this characteristic that makes science both interesting and dynamic. As a part of this continuum, it became possible to create recombinant DNA molecules. Immediately it was recognized that there was a potential for serious adverse events associated with this new technology. Following two scientific conferences at Asilomar, California, the National Institutes of Health moved quickly to create the Recombinant DNA Advisory Committee (RAC). For approximately 38 years the RAC has served as an open forum for review of various recombinant DNA experiments, and for the last 23 years it has played a pivotal role in the oversight of human gene therapy. The RAC's existence obviated the need for more restrictive governmental legislation and has supported the development of genetic interventions that are leading to actual human therapies.

  19. Fine-Structure Mapping of Meiosis-Specific Double-Strand DNA Breaks at a Recombination Hotspot Associated with an Insertion of Telomeric Sequences Upstream of the His4 Locus in Yeast

    PubMed Central

    Xu, F.; Petes, T. D.

    1996-01-01

    Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand DNA breaks (DSBs). Using two approaches, we mapped the position of DSBs associated with a recombination hotspot created by insertion of telomeric sequences into the region upstream of HIS4. We found that the breaks have no obvious sequence specificity and localize to a region of ~50 bp adjacent to the telomeric insertion. By mapping the breaks and by studies of the exonuclease III sensitivity of the broken ends, we conclude that most of the broken DNA molecules have blunt ends with 3'-hydroxyl groups. PMID:8807286

  20. Single Molecule Study of DNA Organization and Recombination

    NASA Astrophysics Data System (ADS)

    Xiao, Botao

    We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

  1. Characterization of recombinant malarial RecQ DNA helicase.

    PubMed

    Suntornthiticharoen, Pattra; Srila, Witsanu; Chavalitshewinkoon-Petmitr, Porntip; Limudomporn, Paviga; Yamabhai, Montarop

    2014-08-01

    RecQ DNA gene of multi-drug resistant Plasmodium falciparum K1 (PfRecQ1) was cloned, and the recombinant C-terminal-decahistidine-tagged PfRecQ1 was expressed in Escherichia coli. The purified enzyme could efficiently unwind partial duplex DNA substrate in a 3' to 5' direction. The malarial RecQ1 could not unwind substrates with both 5' and 3' overhangs, those with a 5' overhang, or blunt-ended DNA duplexes. Unwinding of DNA helicase activity was driven by the hydrolysis of ATP. The drug inhibitory effects of six compounds indicated that only doxorubicin and daunorubicin could inhibit the unwinding activity.

  2. Genome-Wide Analyses of Recombination Prone Regions Predict Role of DNA Structural Motif in Recombination

    PubMed Central

    Das, Swapan Kumar; Chowdhury, Shantanu

    2009-01-01

    HapMap findings reveal surprisingly asymmetric distribution of recombinogenic regions. Short recombinogenic regions (hotspots) are interspersed between large relatively non-recombinogenic regions. This raises the interesting possibility of DNA sequence and/or other cis- elements as determinants of recombination. We hypothesized the involvement of non-canonical sequences that can result in local non-B DNA structures and tested this using the G-quadruplex DNA as a model. G-quadruplex or G4 DNA is a unique form of four-stranded non-B DNA structure that engages certain G-rich sequences, presence of such motifs has been noted within telomeres. In support of this hypothesis, genome-wide computational analyses presented here reveal enrichment of potential G4 (PG4) DNA forming sequences within 25618 human hotspots relative to 9290 coldspots (p<0.0001). Furthermore, co-occurrence of PG4 DNA within several short sequence elements that are associated with recombinogenic regions was found to be significantly more than randomly expected. Interestingly, analyses of more than 50 DNA binding factors revealed that co-occurrence of PG4 DNA with target DNA binding sites of transcription factors c-Rel, NF-kappa B (p50 and p65) and Evi-1 was significantly enriched in recombination-prone regions. These observations support involvement of G4 DNA in recombination, predicting a functional model that is consistent with duplex-strand separation induced by formation of G4 motifs in supercoiled DNA and/or when assisted by other cellular factors. PMID:19198658

  3. A novel DNA selection and direct extraction process and its application in DNA recombination.

    PubMed

    Wang, An-Bang; Cheng, Chia-Wei; Lin, I-Chun; Lu, Fei-Yau; Tsai, Huai-Jen; Lin, Chiu-Chun; Yang, Chun-Hui; Pan, Po-Ting; Kuan, Chen-Chi; Chen, Yen-Chih; Lin, Yi-Wei; Chang, Chih-Ning; Wu, Yi-Hung; Kurniawan, Tetuko; Lin, Chii-Wann; Wo, Andrew M; Chen, Lin-Chi

    2011-02-01

    In the conventional bench-top approach, the DNA recombination process is time- and effort-consuming due to laborious procedures lasting from several hours to a day. A novel DNA selection and direct extraction process has been proposed, integrated and tested on chip. The integrative microfluidic chip can perform the whole procedure of DNA recombination, including DNA digestion, gel electrophoresis, DNA extraction and insert-vector ligation within 1 h. In this high-throughput design, the manual gel cutting was replaced by an automatic processing system that performed high-quality and high-recovery efficiency in DNA extraction process. With no need of gel-dissolving reagents and manipulation, the application of selection and direct extraction process could significantly eliminate the risks from UV and EtBr and also facilitate DNA recombination. Reliable output with high success rate of cloning has been achieved with a significant reduction in operational hazards, required materials, efforts and time.

  4. A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events

    PubMed Central

    Martín-Castellanos, Cristina; Blanco, Miguel; Rozalén, Ana E.; Pérez-Hidalgo, Livia; García, Ana I.; Conde, Francisco; Mata, Juan; Ellermeier, Chad; Davis, Luther; San-Segundo, Pedro; Smith, Gerald R.; Moreno, Sergio

    2009-01-01

    Summary Meiosis is a specialized form of cell division by which sexually reproducing diploid organisms generate haploid gametes. During a long prophase, telomeres cluster into the bouquet configuration to aid chromosome pairing, and DNA replication is followed by high levels of recombination between homologous chromosomes (homologs). This recombination is important for the reductional segregation of homologs at the first meiotic division; without further replication a second meiotic division yields haploid nuclei. In the fission yeast Schizosaccharomyces pombe we have deleted 175 meiotically upregulated genes and found seven genes not previously reported to be critical for meiotic events. Three mutants (rec24, rec25, and rec27) had strongly reduced meiosis-specific DNA double-strand breakage and recombination. One mutant (tht2) was deficient in karyogamy, and two (bqt1 and bqt2) in telomere clustering, explaining their defects in recombination and segregation. The moa1 mutant was delayed in premeiotic S-phase progression and nuclear divisions. Further analysis of these mutants will help elucidate the complex machinery governing the special behavior of meiotic chromosomes. PMID:16303567

  5. A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events.

    PubMed

    Martín-Castellanos, Cristina; Blanco, Miguel; Rozalén, Ana E; Pérez-Hidalgo, Livia; García, Ana I; Conde, Francisco; Mata, Juan; Ellermeier, Chad; Davis, Luther; San-Segundo, Pedro; Smith, Gerald R; Moreno, Sergio

    2005-11-22

    Meiosis is a specialized form of cell division by which sexually reproducing diploid organisms generate haploid gametes. During a long prophase, telomeres cluster into the bouquet configuration to aid chromosome pairing, and DNA replication is followed by high levels of recombination between homologous chromosomes (homologs). This recombination is important for the reductional segregation of homologs at the first meiotic division; without further replication, a second meiotic division yields haploid nuclei. In the fission yeast Schizosaccharomyces pombe, we have deleted 175 meiotically upregulated genes and found seven genes not previously reported to be critical for meiotic events. Three mutants (rec24, rec25, and rec27) had strongly reduced meiosis-specific DNA double-strand breakage and recombination. One mutant (tht2) was deficient in karyogamy, and two (bqt1 and bqt2) were deficient in telomere clustering, explaining their defects in recombination and segregation. The moa1 mutant was delayed in premeiotic S phase progression and nuclear divisions. Further analysis of these mutants will help elucidate the complex machinery governing the special behavior of meiotic chromosomes.

  6. Homologous recombination maintenance of genome integrity during DNA damage tolerance

    PubMed Central

    Prado, Félix

    2014-01-01

    The DNA strand exchange protein Rad51 provides a safe mechanism for the repair of DNA breaks using the information of a homologous DNA template. Homologous recombination (HR) also plays a key role in the response to DNA damage that impairs the advance of the replication forks by providing mechanisms to circumvent the lesion and fill in the tracks of single-stranded DNA that are generated during the process of lesion bypass. These activities postpone repair of the blocking lesion to ensure that DNA replication is completed in a timely manner. Experimental evidence generated over the last few years indicates that HR participates in this DNA damage tolerance response together with additional error-free (template switch) and error-prone (translesion synthesis) mechanisms through intricate connections, which are presented here. The choice between repair and tolerance, and the mechanism of tolerance, is critical to avoid increased mutagenesis and/or genome rearrangements, which are both hallmarks of cancer. PMID:27308329

  7. Cis-effects on Meiotic Recombination Across Distinct a1-sh2 Intervals in a Common Zea Genetic Background

    PubMed Central

    Yao, Hong; Schnable, Patrick S.

    2005-01-01

    Genetic distances across the a1-sh2 interval varied threefold in three near-isogenic stocks that carry structurally distinct teosinte A1 Sh2 haplotypes (from Z. mays spp. mexicana Chalco, Z. mays spp. parviglumis, and Z. luxurians) and a common maize a1::rdt sh2 haplotype. In each haplotype >85% of recombination events resolved in the proximal 10% of the ∼130-kb a1-sh2 interval. Even so, significant differences in the distributions of recombination breakpoints were observed across subintervals among haplotypes. Each of the three previously detected recombination hot spots was detected in at least one of the three teosinte haplotypes and two of these hot spots were not detected in at least one teosinte haplotype. Moreover, novel hot spots were detected in two teosinte haplotypes. Due to the near-isogenic nature of the three stocks, the observed variation in the distribution of recombination events is the consequence of cis-modifications. Although generally negatively correlated with rates of recombination per megabase, levels of sequence polymorphisms do not fully account for the nonrandom distribution of recombination breakpoints. This study also suggests that estimates of linkage disequilibrium must be interpreted with caution when considering whether a gene has been under selection. PMID:15937141

  8. Preferential recombination between GC clusters in yeast mitochondrial DNA.

    PubMed Central

    Dieckmann, C L; Gandy, B

    1987-01-01

    Yeast mitochondrial DNA molecules have long, AT-rich intergenic spacers punctuated by short GC clusters. GC-rich elements have previously been characterized by others as preferred sites for intramolecular recombination leading to the formation of subgenomic petite molecules. In the present study we show that GC clusters are favored sites for intermolecular recombination between a petite and the wild-type grande genome. The petite studied retains 6.5 kb of mitochondrial DNA reiterated tandemly to form molecules consisting of repeated units. Genetic selection for integration of tandem 6.5 kb repeats of the petite into the grande genome yielded a novel recombination event. One of two crossovers in a double exchange event occurred as expected in the 6.5 kb of matching sequence between the genomes, whereas the second exchange involved a 44 bp GC cluster in the petite and another 44 bp GC cluster in the grande genome 700 bp proximal to the region of homology. Creation of a mitochondrial DNA molecule with a repetitive region led to secondary recombination events that generated a family of molecules with zero to several petite units. The finding that 44 bp GC clusters are preferred as sites for intermolecular exchange adds to the data on petite excision implicating these elements as recombinational hotspots in the yeast mitochondrial genome. Images Fig. 3. Fig. 4. Fig. 5. PMID:3327690

  9. The Evolution of Tandemly Repetitive DNA: Recombination Rules

    PubMed Central

    Harding, R. M.; Boyce, A. J.; Clegg, J. B.

    1992-01-01

    Variable numbers of tandem repeats (VNTRs), which include hypervariable regions, minisatellites and microsatellites, can be assigned together with satellite DNAs to define a class of noncoding tandemly repetitive DNA (TR-DNA). The evolution of TR-DNA is assumed to be driven by an unbiased recombinational process. A simulation model of unequal exchange is presented and used to investigate the evolutionary persistence of single TR-DNA lineages. Three different recombination rules are specified to govern the expansion and contraction of a TR-DNA lineage from an initial array of two repeats to, finally, a single repeat allele, which cannot participate in a misalignment and exchange process. In the absence of amplification or selection acting to bias array evolution toward expansion, the probability of attaining a target array size is a function only of the initial number of repeats. We show that the proportions of lineages attaining a targeted array size are the same irrespective of recombination rule and rate, demonstrating that our simulation model is well behaved. The time taken to attain a target array size, the persistence of the target array, and the total persistence time of repetitive array structure, are functions of the initial number of repeats, the rate of recombination, and the rules of misalignment preceding recombinational exchange. These relationships are investigated using our simulation model. While misalignment contraint is probably greatest for satellite DNA it also seems important in accounting for the evolution of VNTR loci including minisatellites. This conclusion is consistent with the observed nonrandom distributions of VNTRs and other TR-DNAs in the human genome. PMID:1468634

  10. A Collaborative, Investigative Recombinant DNA Technology Course with Laboratory

    ERIC Educational Resources Information Center

    Pezzementi, Leo; Johnson, Joy F.

    2002-01-01

    A recombinant DNA technology course was designed to promote contextual, collaborative, inquiry-based learning of science where students learn from one another and have a sense of ownership of their education. The class stressed group presentations and critical reading and discussion of scientific articles. The laboratory consisted of two research…

  11. Human DNA repair and recombination genes

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

  12. Chromosome choreography: the meiotic ballet.

    PubMed

    Page, Scott L; Hawley, R Scott

    2003-08-08

    The separation of homologous chromosomes during meiosis in eukaryotes is the physical basis of Mendelian inheritance. The core of the meiotic process is a specialized nuclear division (meiosis I) in which homologs pair with each other, recombine, and then segregate from each other. The processes of chromosome alignment and pairing allow for homolog recognition. Reciprocal meiotic recombination ensures meiotic chromosome segregation by converting sister chromatid cohesion into mechanisms that hold homologous chromosomes together. Finally, the ability of sister kinetochores to orient to a single pole at metaphase I allows the separation of homologs to two different daughter cells. Failures to properly accomplish this elegant chromosome dance result in aneuploidy, a major cause of miscarriage and birth defects in human beings.

  13. Role of Recombinant DNA Technology to Improve Life

    PubMed Central

    Khan, Suliman; Ullah, Muhammad Wajid; Siddique, Rabeea; Nabi, Ghulam; Manan, Sehrish; Yousaf, Muhammad

    2016-01-01

    In the past century, the recombinant DNA technology was just an imagination that desirable characteristics can be improved in the living bodies by controlling the expressions of target genes. However, in recent era, this field has demonstrated unique impacts in bringing advancement in human life. By virtue of this technology, crucial proteins required for health problems and dietary purposes can be produced safely, affordably, and sufficiently. This technology has multidisciplinary applications and potential to deal with important aspects of life, for instance, improving health, enhancing food resources, and resistance to divergent adverse environmental effects. Particularly in agriculture, the genetically modified plants have augmented resistance to harmful agents, enhanced product yield, and shown increased adaptability for better survival. Moreover, recombinant pharmaceuticals are now being used confidently and rapidly attaining commercial approvals. Techniques of recombinant DNA technology, gene therapy, and genetic modifications are also widely used for the purpose of bioremediation and treating serious diseases. Due to tremendous advancement and broad range of application in the field of recombinant DNA technology, this review article mainly focuses on its importance and the possible applications in daily life. PMID:28053975

  14. Role of Recombinant DNA Technology to Improve Life.

    PubMed

    Khan, Suliman; Ullah, Muhammad Wajid; Siddique, Rabeea; Nabi, Ghulam; Manan, Sehrish; Yousaf, Muhammad; Hou, Hongwei

    2016-01-01

    In the past century, the recombinant DNA technology was just an imagination that desirable characteristics can be improved in the living bodies by controlling the expressions of target genes. However, in recent era, this field has demonstrated unique impacts in bringing advancement in human life. By virtue of this technology, crucial proteins required for health problems and dietary purposes can be produced safely, affordably, and sufficiently. This technology has multidisciplinary applications and potential to deal with important aspects of life, for instance, improving health, enhancing food resources, and resistance to divergent adverse environmental effects. Particularly in agriculture, the genetically modified plants have augmented resistance to harmful agents, enhanced product yield, and shown increased adaptability for better survival. Moreover, recombinant pharmaceuticals are now being used confidently and rapidly attaining commercial approvals. Techniques of recombinant DNA technology, gene therapy, and genetic modifications are also widely used for the purpose of bioremediation and treating serious diseases. Due to tremendous advancement and broad range of application in the field of recombinant DNA technology, this review article mainly focuses on its importance and the possible applications in daily life.

  15. Evolutionary conservation of meiotic DSB proteins: more than just Spo11.

    PubMed

    Cole, Francesca; Keeney, Scott; Jasin, Maria

    2010-06-15

    Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs) generated by the Spo11 protein. In budding yeast, five other meiotic-specific proteins are also required for DSB formation, but, with rare exception, orthologs had not been identified in other species. In this issue of Genes & Development, Kumar and colleagues (pp. 1266-1280) used a phylogenomic approach to identify two of these proteins across multiple clades, and confirmed that one of these, MEI4, is a functional ortholog in mouse.

  16. Meiotic abnormalities

    SciTech Connect

    1993-12-31

    Chapter 19, describes meiotic abnormalities. These include nondisjunction of autosomes and sex chromosomes, genetic and environmental causes of nondisjunction, misdivision of the centromere, chromosomally abnormal human sperm, male infertility, parental age, and origin of diploid gametes. 57 refs., 2 figs., 1 tab.

  17. Direct facile screening of recombinant DNA vector constructs.

    PubMed

    Winnard, Paul T; Challa, Rushi; Bhujwalla, Zaver M; Raman, Venu

    2014-04-01

    Direct efficient facile screening of bacterial transformants with the goal of selecting, retrieving, and using recombinant DNA is exemplified by simple visual-based colorimetric inspections or fluorescent protein-based assays. We describe pRedScript, which introduces the constitutive expression of a very bright red fluorescent protein into transformants. On agar plates, red colonies are simply visualized in ambient white light in stark contrast to recombinant transformants that are white. In addition, the bright red fluorescence of the reporter protein can also be harnessed as a sensitive signal for screening bacterial promoters during the development of optimized fermentation conditions.

  18. Endonucleases involved in repair and recombination of DNA

    SciTech Connect

    Linn, S.M.

    1988-01-01

    When our DOE support began as a contract in 1970, from the AEC, it was our intent to begin to understand how several enzymes which we had detected in E. coli might be involved in DNA recombination and repair. These studies led to our characterization of the recBC DNase (exonuclease 5) as well as endonucleases 3 and 5. As research supported by that contract progressed, we expanded our interests to include mammalian enzymes involved in base excision repair, most notably AP endonucleases, DNA glycosylases and DNA purine insertase. A logical next step involved the inclusion of DNA polymerases into our studies of repair. Current progress includes research on: isolation of xeroderma pigmentosum correction factors; isolation of ultraviolet (UV) endonucleases; mitochondrial repair enzymes; alkylation damage repair; comparisons of repair in normal diploid, transformed, and non-mitotic cells; and repair reactions by DNA polymerases.

  19. 76 FR 62816 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-11

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines... recombinant DNA research. OBA is also specifying the risk group for several viruses not previously listed...

  20. 75 FR 31795 - Office of Biotechnology Activities; Recombinant DNA Research: Amended Notice of Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-04

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA... ] under Section III-A-1 of the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH... the NIH Recombinant DNA Advisory Committee has been deferred at the request of the...

  1. 76 FR 44339 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-25

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines... attenuated strains of bacteria and viruses that are frequently used in recombinant DNA research. OBA is...

  2. 75 FR 69687 - Office of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-15

    ... of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) ACTION: Notice of consideration of proposed...- vector system may be certified only after review by the NIH Recombinant DNA Advisory Committee (RAC)...

  3. 76 FR 27653 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-12

    ... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines... Kluyveromyces lactis as a host-vector 1 system has been reviewed by the NIH ] Recombinant DNA Advisory...

  4. Analysis of chromatin structure at meiotic DSB sites in yeasts.

    PubMed

    Hirota, Kouji; Fukuda, Tomoyuki; Yamada, Takatomi; Ohta, Kunihiro

    2009-01-01

    One of the major features of meiosis is a high frequency of homologous recombination that not only confers genetic diversity to a successive generation but also ensures proper segregation of chromosomes. Meiotic recombination is initiated by DNA double-strand breaks that require many proteins including the catalytic core, Spo11. In this regard, like transcription and repair, etc., recombination is hindered by a compacted chromatin structure because trans-acting factors cannot easily access the DNA. Such inhibitory effects must be alleviated prior to recombination initiation. Indeed, a number of groups showed that chromatin around recombination hotspots is less condensed, by using nucleases as a probe to assess local DNA accessibility. Here we describe a method to analyze chromatin structure of a recombination hotspot in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This method, combining micrococcal nuclease (MNase) digestion ofchromatin DNA and subsequent Southern blotting, is expected to provide information as to chromatin context around a hotspot. Moreover, by virtue of MNase preferentially targeting linker DNA, positions of several nucleosomes surrounding a hotspot can also be determined. Our protocol is a very powerful way to analyze several-kb regions of interest and can be applied to other purposes.

  5. Genome Evolution and Meiotic Maps by Massively Parallel DNA Sequencing: Spotted Gar, an Outgroup for the Teleost Genome Duplication

    PubMed Central

    Amores, Angel; Catchen, Julian; Ferrara, Allyse; Fontenot, Quenton; Postlethwait, John H.

    2011-01-01

    Genomic resources for hundreds of species of evolutionary, agricultural, economic, and medical importance are unavailable due to the expense of well-assembled genome sequences and difficulties with multigenerational studies. Teleost fish provide many models for human disease but possess anciently duplicated genomes that sometimes obfuscate connectivity. Genomic information representing a fish lineage that diverged before the teleost genome duplication (TGD) would provide an outgroup for exploring the mechanisms of evolution after whole-genome duplication. We exploited massively parallel DNA sequencing to develop meiotic maps with thrift and speed by genotyping F1 offspring of a single female and a single male spotted gar (Lepisosteus oculatus) collected directly from nature utilizing only polymorphisms existing in these two wild individuals. Using Stacks, software that automates the calling of genotypes from polymorphisms assayed by Illumina sequencing, we constructed a map containing 8406 markers. RNA-seq on two map-cross larvae provided a reference transcriptome that identified nearly 1000 mapped protein-coding markers and allowed genome-wide analysis of conserved synteny. Results showed that the gar lineage diverged from teleosts before the TGD and its genome is organized more similarly to that of humans than teleosts. Thus, spotted gar provides a critical link between medical models in teleost fish, to which gar is biologically similar, and humans, to which gar is genomically similar. Application of our F1 dense mapping strategy to species with no prior genome information promises to facilitate comparative genomics and provide a scaffold for ordering the numerous contigs arising from next generation genome sequencing. PMID:21828280

  6. ATM controls meiotic double-strand-break formation.

    PubMed

    Lange, Julian; Pan, Jing; Cole, Francesca; Thelen, Michael P; Jasin, Maria; Keeney, Scott

    2011-10-16

    In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation. However, improperly repaired DSBs can cause meiotic arrest or mutation; thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse and SPO11 and its accessory factors remain abundant long after most DSB formation ceases, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency.

  7. Jeremy Rifkin challenges recombinant DNA research: A rhetoric of heresy

    SciTech Connect

    Futrell, W.M.

    1992-01-01

    One significant issue to come before the public in recent years is recombinant DNA research or genetic engineering and its applications. An important spokesman on this issue is Jeremy Rifkin. Rifkin is of rhetorical interest because of his strategies to sustain the dialogue and define the parameters in which it occurs. This dissertation analyzes a broad range of Rifkin's rhetorical artifacts and those of scientists engaged in recombinant DNA research. They are examined against criteria developed to identify and understand heresy. The five areas of analysis are: the nearness/remoteness phenomenon, the social construction of heresy, the social consequences of heresy, the doctrinal consequences of heresy, and the heresy-hunt ritual. The first two criteria focus on the rhetorical strategies of the heretic. The last three concentrate on the rhetorical strategies of the defenders of the institutional orthodoxy. This dissertation examines the rhetorical strategies of a heretical challenge to the scientific establishment and the consequences of that challenge. This dissertation also analyzes the rhetorical strategies employed by the defenders of the scientific orthodoxy. Although an understanding of the rhetorical strategies employed on both sides of this conflict is important, the implications for the role of rhetoric in highly controversial issues such as recombinant DNA are even more critical.

  8. Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription.

    PubMed Central

    Kogoma, T

    1997-01-01

    Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored. PMID:9184011

  9. The case of the fickle fingers: how the PRDM9 zinc finger protein specifies meiotic recombination hotspots in humans.

    PubMed

    Ségurel, Laure; Leffler, Ellen Miranda; Przeworski, Molly

    2011-12-01

    During mammalian meiosis, double-strand breaks are deliberately made throughout the genome and then repaired, leading to the exchange of genetic material between copies of chromosomes. How the locations of breaks are specified was largely unknown until a fortuitous confluence of statistical genetics and molecular biology uncovered the role of PRDM9, a DNA binding protein. Many properties of this protein remain mysterious, however, including how it binds to DNA, how it contributes to male infertility-both in humans, and in hybrid mice-and why, in spite of its fundamental function in meiosis, its binding domain varies extensively among humans and across mammals. We present a brief summary of what has recently been learned about PRDM9 in different fields, focusing on the puzzles yet to be resolved.

  10. Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

    SciTech Connect

    Not Available

    1984-01-01

    This volume contains full papers prepared by the participants to the 1984 Cold Springs Harbor Symposia on Quantitative Biology. This year's theme is entitled Recombination at the DNA level. The volume consists of 93 articles grouped into subject areas entitled chromosome mechanics, yeast systems, mammalian homologous recombination, transposons, mu, plant transposons/T4 recombination, topoisomerase, resolvase and gyrase, Escherichia coli general recombination, RecA, repair, leukaryotic enzymes, integration and excision of bacteriophage, site-specific recombination, and recombination in vitro.

  11. Successive site translocating inoculation potentiates DNA/recombinant vaccinia vaccination

    PubMed Central

    Ren, Yanqin; Wang, Na; Hu, Weiguo; Zhang, Xiaoyan; Xu, Jianqing; Wan, Yanmin

    2015-01-01

    DNA vaccines have advantages over traditional vaccine modalities; however the relatively low immunogenicity restrains its translation into clinical use. Further optimizations are needed to get the immunogenicity of DNA vaccine closer to the level required for human use. Here we show that intramuscularly inoculating into a different limb each time significantly improves the immunogenicities of both DNA and recombinant vaccinia vaccines during multiple vaccinations, compared to repeated vaccination on the same limb. We term this strategy successive site translocating inoculation (SSTI). SSTI could work in synergy with genetic adjuvant and DNA prime-recombinant vaccinia boost regimen. By comparing in vivo antigen expression, we found that SSTI avoided the specific inhibition of in vivo antigen expression, which was observed in the limbs being repeatedly inoculated. Employing in vivo T cell depletion and passive IgG transfer, we delineated that the inhibition was not mediated by CD8+ T cells but by specific antibodies. Finally, by using C3−/− mouse model and in vivo NK cells depletion, we identified that specific antibodies negatively regulated the in vivo antigen expression primarily in a complement depended way. PMID:26667202

  12. Successive site translocating inoculation potentiates DNA/recombinant vaccinia vaccination.

    PubMed

    Ren, Yanqin; Wang, Na; Hu, Weiguo; Zhang, Xiaoyan; Xu, Jianqing; Wan, Yanmin

    2015-12-15

    DNA vaccines have advantages over traditional vaccine modalities; however the relatively low immunogenicity restrains its translation into clinical use. Further optimizations are needed to get the immunogenicity of DNA vaccine closer to the level required for human use. Here we show that intramuscularly inoculating into a different limb each time significantly improves the immunogenicities of both DNA and recombinant vaccinia vaccines during multiple vaccinations, compared to repeated vaccination on the same limb. We term this strategy successive site translocating inoculation (SSTI). SSTI could work in synergy with genetic adjuvant and DNA prime-recombinant vaccinia boost regimen. By comparing in vivo antigen expression, we found that SSTI avoided the specific inhibition of in vivo antigen expression, which was observed in the limbs being repeatedly inoculated. Employing in vivo T cell depletion and passive IgG transfer, we delineated that the inhibition was not mediated by CD8(+) T cells but by specific antibodies. Finally, by using C3(-/-) mouse model and in vivo NK cells depletion, we identified that specific antibodies negatively regulated the in vivo antigen expression primarily in a complement depended way.

  13. Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

    PubMed Central

    Deng, W P; Nickoloff, J A

    1994-01-01

    Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication. Images PMID:8264607

  14. A role for the malignant brain tumour (MBT) domain protein LIN-61 in DNA double-strand break repair by homologous recombination.

    PubMed

    Johnson, Nicholas M; Lemmens, Bennie B L G; Tijsterman, Marcel

    2013-01-01

    Malignant brain tumour (MBT) domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR) for the repair of DNA double-strand breaks (DSBs). lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT-deficient tumours may also have defective DSB repair.

  15. Choreography of recombination proteins during the DNA damage response.

    PubMed

    Lisby, Michael; Rothstein, Rodney

    2009-09-02

    Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such genetic alterations are the main causes of cancer and other genetic diseases. Consequently, DNA double-strand break repair (DSBR) is an important process in all living organisms. DSBR is also the driving mechanism in most strategies of gene targeting, which has applications in both genetic and clinical research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells.

  16. Meiotic Development in Caenorhabditis elegans

    PubMed Central

    Lui, Doris Y.

    2013-01-01

    Caenorhabditis elegans has become a powerful experimental organism with which to study meiotic processes that promote the accurate segregation of chromosomes during the generation of haploid gametes. Haploid reproductive cells are produced through one round of chromosome replication followed by two successive cell divisions. Characteristic meiotic chromosome structure and dynamics are largely conserved in C. elegans. Chromosomes adopt a meiosis-specific structure by loading cohesin proteins, assembling axial elements, and acquiring chromatin marks. Homologous chromosomes pair and form physical connections though synapsis and recombination. Synaptonemal complex and crossover formation allow for the homologs to stably associate prior to remodeling that facilitates their segregation. This chapter will cover conserved meiotic processes as well as highlight aspects of meiosis that are unique to C. elegans. PMID:22872477

  17. Measuring Meiotic Crossovers via Multi-Locus Genotyping of Single Pollen Grains in Barley.

    PubMed

    Dreissig, Steven; Fuchs, Jörg; Cápal, Petr; Kettles, Nicola; Byrne, Ed; Houben, Andreas

    2015-01-01

    The detection of meiotic crossovers in crop plants currently relies on scoring DNA markers in a segregating population or cytological visualization. We investigated the feasibility of using flow-sorted haploid nuclei, Phi29 DNA polymerase-based whole-genome-amplification (WGA) and multi-locus KASP-genotyping to measure meiotic crossovers in individual barley pollen grains. To demonstrate the proof of concept, we used 24 gene-based physically mapped single nucleotide polymorphisms to genotype the WGA products of 50 single pollen nuclei. The number of crossovers per chromosome, recombination frequencies along chromosome 3H and segregation distortion were analysed and compared to a doubled haploid (DH) population of the same genotype. The number of crossovers and chromosome wide recombination frequencies show that this approach is able to produce results that resemble those obtained from other methods in a biologically meaningful way. Only the segregation distortion was found to be lower in the pollen population than in DH plants.

  18. Recombinant methods for screening human DNA excision repair proficiency

    SciTech Connect

    Athas, W.F.

    1988-01-01

    A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period.

  19. DNA replication meets genetic exchange: chromosomal damage and its repair by homologous recombination.

    PubMed

    Kuzminov, A

    2001-07-17

    Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.

  20. Overlapping mechanisms promote postsynaptic RAD-51 filament disassembly during meiotic double-strand break repair.

    PubMed

    Ward, Jordan D; Muzzini, Diego M; Petalcorin, Mark I R; Martinez-Perez, Enrique; Martin, Julie S; Plevani, Paolo; Cassata, Giuseppe; Marini, Federica; Boulton, Simon J

    2010-01-29

    Homologous recombination (HR) is essential for repair of meiotic DNA double-strand breaks (DSBs). Although the mechanisms of RAD-51-DNA filament assembly and strand exchange are well characterized, the subsequent steps of HR are less well defined. Here, we describe a synthetic lethal interaction between the C. elegans helicase helq-1 and RAD-51 paralog rfs-1, which results in a block to meiotic DSB repair after strand invasion. Whereas RAD-51-ssDNA filaments assemble at meiotic DSBs with normal kinetics in helq-1, rfs-1 double mutants, persistence of RAD-51 foci and genetic interactions with rtel-1 suggest a failure to disassemble RAD-51 from strand invasion intermediates. Indeed, purified HELQ-1 and RFS-1 independently bind to and promote the disassembly of RAD-51 from double-stranded, but not single-stranded, DNA filaments via distinct mechanisms in vitro. These results indicate that two compensating activities are required to promote postsynaptic RAD-51 filament disassembly, which are collectively essential for completion of meiotic DSB repair.

  1. Process of labeling specific chromosomes using recombinant repetitive DNA

    DOEpatents

    Moyzis, R.K.; Meyne, J.

    1988-02-12

    Chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family members and consensus sequences of the repetitive DNA families for the chromosome preferential sequences. The selected low homology regions are then hybridized with chromosomes to determine those low homology regions hybridized with a specific chromosome under normal stringency conditions.

  2. Meiotic DSB patterning: A multifaceted process.

    PubMed

    Cooper, Tim J; Garcia, Valerie; Neale, Matthew J

    2016-01-01

    Meiosis is a specialized two-step cell division responsible for genome haploidization and the generation of genetic diversity during gametogenesis. An integral and distinctive feature of the meiotic program is the evolutionarily conserved initiation of homologous recombination (HR) by the developmentally programmed induction of DNA double-strand breaks (DSBs). The inherently dangerous but essential act of DSB formation is subject to multiple forms of stringent and self-corrective regulation that collectively ensure fruitful and appropriate levels of genetic exchange without risk to cellular survival. Within this article we focus upon an emerging element of this control--spatial regulation--detailing recent advances made in understanding how DSBs are evenly distributed across the genome, and present a unified view of the underlying patterning mechanisms employed.

  3. Meiotic DSB patterning: A multifaceted process

    PubMed Central

    Cooper, Tim J.; Garcia, Valerie; Neale, Matthew J.

    2016-01-01

    Abstract Meiosis is a specialized two-step cell division responsible for genome haploidization and the generation of genetic diversity during gametogenesis. An integral and distinctive feature of the meiotic program is the evolutionarily conserved initiation of homologous recombination (HR) by the developmentally programmed induction of DNA double-strand breaks (DSBs). The inherently dangerous but essential act of DSB formation is subject to multiple forms of stringent and self-corrective regulation that collectively ensure fruitful and appropriate levels of genetic exchange without risk to cellular survival. Within this article we focus upon an emerging element of this control—spatial regulation—detailing recent advances made in understanding how DSBs are evenly distributed across the genome, and present a unified view of the underlying patterning mechanisms employed. PMID:26730703

  4. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  5. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  6. Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break.

    PubMed

    Brown, M Scott; Grubb, Jennifer; Zhang, Annie; Rust, Michael J; Bishop, Douglas K

    2015-12-01

    The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs). Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments.

  7. Analysis and optimization of recombinant DNA joining reactions.

    PubMed

    Legerski, R J; Robberson, D L

    1985-01-20

    The statistical segment length of duplex DNA was determined in phage T4 ligase (poly(deoxyribonucleotide): poly(deoxyribonucleotide) ligase (AMP forming), EC 6.5.1.1) buffer (50 mM-Tris . HCl (pH 7.8), 20 mM-dithiothreitol, 10 mM-MgCl2, 1 mM-ATP) at 12 degrees C to be 1030(+/- 116) A. This result was obtained by electron microscopic examination of the molecular distributions generated by T4 ligase-mediated joining of EcoRI-cleaved pBR322 DNA. This value of the statistical segment length was utilized in an extension of the Jacobson-Stockmayer theory on the probability of intramolecular cyclization in order to optimize DNA joining reactions that are of great utility in recombinant DNA experiments. Five cloning systems were analyzed: circular plasmid vectors that had been linearized with one or two restriction endonucleases, circular plasmids that had been tailed with deoxyhomopolymers before joining, lambda-type cloning vectors and cosmids. The results are tabulated for convenient use in molecular cloning experiments.

  8. Bacterial DNA repair genes and their eukaryotic homologues: 5. The role of recombination in DNA repair and genome stability.

    PubMed

    Nowosielska, Anetta

    2007-01-01

    Recombinational repair is a well conserved DNA repair mechanism present in all living organisms. Repair by homologous recombination is generally accurate as it uses undamaged homologous DNA molecule as a repair template. In Escherichia coli homologous recombination repairs both the double-strand breaks and single-strand gaps in DNA. DNA double-strand breaks (DSB) can be induced upon exposure to exogenous sources such as ionizing radiation or endogenous DNA-damaging agents including reactive oxygen species (ROS) as well as during natural biological processes like conjugation. However, the bulk of double strand breaks are formed during replication fork collapse encountering an unrepaired single strand gap in DNA. Under such circumstances DNA replication on the damaged template can be resumed only if supported by homologous recombination. This functional cooperation of homologous recombination with replication machinery enables successful completion of genome duplication and faithful transmission of genetic material to a daughter cell. In eukaryotes, homologous recombination is also involved in essential biological processes such as preservation of genome integrity, DNA damage checkpoint activation, DNA damage repair, DNA replication, mating type switching, transposition, immune system development and meiosis. When unregulated, recombination can lead to genome instability and carcinogenesis.

  9. Recombination Promoted by DNA Viruses: Phage λ to Herpes Simplex Virus

    PubMed Central

    Weller, Sandra K.; Sawitzke, James A.

    2015-01-01

    The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies. PMID:25002096

  10. Recombination promoted by DNA viruses: phage λ to herpes simplex virus.

    PubMed

    Weller, Sandra K; Sawitzke, James A

    2014-01-01

    The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies.

  11. Meiotic chromosome mobility in fission yeast is resistant to environmental stress

    PubMed Central

    Illner, Doris; Lorenz, Alexander; Scherthan, Harry

    2016-01-01

    The formation of healthy gametes requires pairing of homologous chromosomes (homologs) as a prerequisite for their correct segregation during meiosis. Initially, homolog alignment is promoted by meiotic chromosome movements feeding into intimate homolog pairing by homologous recombination and/or synaptonemal complex formation. Meiotic chromosome movements in the fission yeast, Schizosaccharomyces pombe, depend on astral microtubule dynamics that drag the nucleus through the zygote; known as horsetail movement. The response of microtubule-led meiotic chromosome movements to environmental stresses such as ionizing irradiation (IR) and associated reactive oxygen species (ROS) is not known. Here, we show that, in contrast to budding yeast, the horsetail movement is largely radiation-resistant, which is likely mediated by a potent antioxidant defense. IR exposure of sporulating S. pombe cells induced misrepair and irreparable DNA double strand breaks causing chromosome fragmentation, missegregation and gamete death. Comparing radiation outcome in fission and budding yeast, and studying meiosis with poisoned microtubules indicates that the increased gamete death after IR is innate to fission yeast. Inhibition of meiotic chromosome mobility in the face of IR failed to influence the course of DSB repair, indicating that paralysis of meiotic chromosome mobility in a genotoxic environment is not a universal response among species. PMID:27074839

  12. SSP1, a gene necessary for proper completion of meiotic divisions and spore formation in Saccharomyces cerevisiae.

    PubMed Central

    Nag, D K; Koonce, M P; Axelrod, J

    1997-01-01

    During meiosis, a diploid cell undergoes two rounds of nuclear division following one round of DNA replication to produce four haploid gametes. In yeast, haploid meiotic products are packaged into spores. To gain new insights into meiotic development and spore formation, we followed differential expression of genes in meiotic versus vegetatively growing cells in the yeast Saccharomyces cerevisiae. Our results indicate that there are at least five different classes of transcripts representing genes expressed at different stages of the sporulation program. Here we describe one of these differentially expressed genes, SSP1, which plays an essential role in meiosis and spore formation. SSP1 is expressed midway through meiosis, and homozygous ssp1 diploid cells fail to sporulate. In the ssp1 mutant, meiotic recombination is normal but viability declines rapidly. Both meiotic divisions occur at the normal time; however, the fraction of cells completing meiosis is significantly reduced, and nuclei become fragmented soon after meiosis II. The ssp1 defect does not appear to be related to a microtubule-cytoskeletal-dependent event and is independent of two rounds of chromosome segregation. The data suggest that Ssp1 is likely to function in a pathway that controls meiotic nuclear divisions and coordinates meiosis and spore formation. PMID:9372934

  13. Statistical Analysis on Detecting Recombination Sites in DNA-β Satellites Associated with Old World Geminiviruses

    PubMed Central

    Xu, Kai; Yoshida, Ruriko

    2010-01-01

    Although exchange of genetic information by recombination plays an important role in the evolution of viruses, it is not clear how it generates diversity. Understanding recombination events helps with the study of the evolution of new virus strains or new viruses. Geminiviruses are plant viruses which have ambisense single-stranded circular DNA genomes and are one of the most economically important plant viruses in agricultural production. Small circular single-stranded DNA satellites, termed DNA-β, have recently been found to be associated with some geminivirus infections. In this paper we analyze several DNA-β sequences of geminiviruses for recombination events using phylogenetic and statistical analysis and we find that one strain from ToLCMaB has a recombination pattern and is a recombinant molecule between two strains from two species, PaLCuB-[IN:Chi:05] (major parent) and ToLCB-[IN:CP:04] (minor parent). We propose that this recombination event contributed to the evolution of the strain of ToLCMaB in South India. The Hidden Markov Chain (HMM) method developed by Webb et al. (2009) estimating phylogenetic tree through out the whole alignment provide us a recombination history of these DNA-β strains. It is the first time that this statistic method has been used on DNArecombination study and give a clear recombination history of DNArecombination. PMID:21423447

  14. Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures

    SciTech Connect

    Chen, Zhucheng; Yang, Haijuan; Pavletich, Nikola P

    2008-07-08

    The RecA family of ATPases mediates homologous recombination, a reaction essential for maintaining genomic integrity and for generating genetic diversity. RecA, ATP and single-stranded DNA (ssDNA) form a helical filament that binds to double-stranded DNA (dsDNA), searches for homology, and then catalyses the exchange of the complementary strand, producing a new heteroduplex. Here we have solved the crystal structures of the Escherichia coli RecA-ssDNA and RecA-heteroduplex filaments. They show that ssDNA and ATP bind to RecA-RecA interfaces cooperatively, explaining the ATP dependency of DNA binding. The ATP {gamma}-phosphate is sensed across the RecA-RecA interface by two lysine residues that also stimulate ATP hydrolysis, providing a mechanism for DNA release. The DNA is underwound and stretched globally, but locally it adopts a B-DNA-like conformation that restricts the homology search to Watson-Crick-type base pairing. The complementary strand interacts primarily through base pairing, making heteroduplex formation strictly dependent on complementarity. The underwound, stretched filament conformation probably evolved to destabilize the donor duplex, freeing the complementary strand for homology sampling.

  15. The Mouse INO80 Chromatin-Remodeling Complex Is an Essential Meiotic Factor for Spermatogenesis1

    PubMed Central

    Serber, Daniel W.; Runge, John S.; Menon, Debashish U.; Magnuson, Terry

    2015-01-01

    The ability to faithfully transmit genetic information across generations via the germ cells is a critical aspect of mammalian reproduction. The process of germ cell development requires a number of large-scale modulations of chromatin within the nucleus. One such occasion arises during meiotic recombination, when hundreds of DNA double-strand breaks are induced and subsequently repaired, enabling the transfer of genetic information between homologous chromosomes. The inability to properly repair DNA damage is known to lead to an arrest in the developing germ cells and sterility within the animal. Chromatin-remodeling activity, and in particular the BRG1 subunit of the SWI/SNF complex, has been shown to be required for successful completion of meiosis. In contrast, remodeling complexes of the ISWI and CHD families are required for postmeiotic processes. Little is known regarding the contribution of the INO80 family of chromatin-remodeling complexes, which is a particularly interesting candidate due to its well described functions during DNA double-strand break repair. Here we show that INO80 is expressed in developing spermatocytes during the early stages of meiotic prophase I. Based on this information, we used a conditional allele to delete the INO80 core ATPase subunit, thereby eliminating INO80 chromatin-remodeling activity in this lineage. The loss of INO80 resulted in an arrest during meiosis associated with a failure to repair DNA damage during meiotic recombination. PMID:26607718

  16. DEK is required for homologous recombination repair of DNA breaks

    PubMed Central

    Smith, Eric A.; Gole, Boris; Willis, Nicholas A.; Soria, Rebeca; Starnes, Linda M.; Krumpelbeck, Eric F.; Jegga, Anil G.; Ali, Abdullah M.; Guo, Haihong; Meetei, Amom R.; Andreassen, Paul R.; Kappes, Ferdinand; Vinnedge, Lisa M. Privette; Daniel, Jeremy A.; Scully, Ralph; Wiesmüller, Lisa; Wells, Susanne I.

    2017-01-01

    DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition. PMID:28317934

  17. DEK is required for homologous recombination repair of DNA breaks.

    PubMed

    Smith, Eric A; Gole, Boris; Willis, Nicholas A; Soria, Rebeca; Starnes, Linda M; Krumpelbeck, Eric F; Jegga, Anil G; Ali, Abdullah M; Guo, Haihong; Meetei, Amom R; Andreassen, Paul R; Kappes, Ferdinand; Vinnedge, Lisa M Privette; Daniel, Jeremy A; Scully, Ralph; Wiesmüller, Lisa; Wells, Susanne I

    2017-03-20

    DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.

  18. Gene expression profiles of Spo11-/- mouse testes with spermatocytes arrested in meiotic prophase I.

    PubMed

    Smirnova, Natalya A; Romanienko, Peter J; Khil, Pavel P; Camerini-Otero, R Daniel

    2006-07-01

    Spo11, a meiosis-specific protein, introduces double-strand breaks on chromosomal DNA and initiates meiotic recombination in a wide variety of organisms. Mouse null Spo11 spermatocytes fail to synapse chromosomes and progress beyond the zygotene stage of meiosis. We analyzed gene expression profiles in Spo11(-/ -)adult and juvenile wild-type testis to describe genes expressed before and after the meiotic arrest resulting from the knocking out of Spo11. These genes were characterized using the Gene Ontology data base. To focus on genes involved in meiosis, we performed comparative gene expression analysis of Spo11(-/ -)and wild-type testes from 15-day mice, when spermatocytes have just entered pachytene. We found that the knockout of Spo11 causes dramatic changes in the level of expression of genes that participate in meiotic recombination (Hop2, Brca2, Mnd1, FancG) and in the meiotic checkpoint (cyclin B2, Cks2), but does not affect genes encoding protein components of the synaptonemal complex. Finally, we discovered unknown genes that are affected by the disruption of the Spo11 gene and therefore may be specifically involved in meiosis and spermatogenesis.

  19. Would Dissociative Recombination of DNA+ be a Possible Pathway of DNA Damage?

    NASA Astrophysics Data System (ADS)

    Kwon, H. C.; Chen, Z. P.; Strom, R. A.; Andrianarijaona, V. M.

    2015-05-01

    It is known that dissociative recombination (DR) is one of the very efficient processes of destruction of molecular cations into neutral particles. During the past few years, the focus of DR has been expanded from small inorganic molecules to macromolecular cation. We are probing the possibility of the DR of DNA+ after ionization of DNA, for example due to ionizing radiation. Therefore we are investigating the existence of autoionization states within nucleotide bases (Guanine, Adenine, Cytosine, and Thymine). Our results from computational analysis using the modern electronic structure program ORCA will be presented. Authors wish to give special thanks to Pacific Union College Student Senate for their financial support.

  20. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is...

  1. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is...

  2. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is...

  3. Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

    PubMed Central

    Kuzminov, Andrei

    1999-01-01

    Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation. PMID:10585965

  4. Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease.

    PubMed

    Yeeles, Joseph T P; van Aelst, Kara; Dillingham, Mark S; Moreno-Herrero, Fernando

    2011-06-24

    AddAB is a helicase-nuclease that processes double-stranded DNA breaks for repair by homologous recombination. This process is modulated by Chi recombination hotspots: specific DNA sequences that attenuate the nuclease activity of the translocating AddAB complex to promote downstream recombination. Using a combination of kinetic and imaging techniques, we show that AddAB translocation is not coupled to DNA unwinding in the absence of single-stranded DNA binding proteins because nascent single-stranded DNA immediately re-anneals behind the moving enzyme. However, recognition of recombination hotspot sequences during translocation activates unwinding by coupling these activities, thereby ensuring the downstream formation of single-stranded DNA that is required for RecA-mediated recombinational repair. In addition to their implications for the mechanism of double-stranded DNA break repair, these observations may affect our implementation and interpretation of helicase assays and our understanding of helicase mechanisms in general.

  5. Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton

    PubMed Central

    Illner, Doris; Scherthan, Harry

    2013-01-01

    Meiosis generates haploid cells or spores for sexual reproduction. As a prelude to haploidization, homologous chromosomes pair and recombine to undergo segregation during the first meiotic division. During the entire meiotic prophase of the yeast Saccharomyces cerevisiae, chromosomes perform rapid movements that are suspected to contribute to the regulation of recombination. Here, we investigated the impact of ionizing radiation (IR) on movements of GFP–tagged bivalents in live pachytene cells. We find that exposure of sporulating cultures with >40 Gy (4-krad) X-rays stalls pachytene chromosome movements. This identifies a previously undescribed acute radiation response in yeast meiosis, which contrasts with its reported radioresistance of up to 1,000 Gy in survival assays. A modified 3′-end labeling assay disclosed IR-induced dsDNA breaks (DSBs) in pachytene cells at a linear dose relationship of one IR-induced DSB per cell per 5 Gy. Dihydroethidium staining revealed formation of reactive oxygen species (ROS) in irradiated cells. Immobility of fuzzy-appearing irradiated bivalents was rescued by addition of radical scavengers. Hydrogen peroxide-induced ROS did reduce bivalent mobility similar to 40 Gy X IR, while they failed to induce DSBs. IR- and H2O2-induced ROS were found to decompose actin cables that are driving meiotic chromosome mobility, an effect that could be rescued by antioxidant treatment. Hence, it appears that the meiotic actin cytoskeleton is a radical-sensitive system that inhibits bivalent movements in response to IR- and oxidant-induced ROS. This may be important to prevent motility-driven unfavorable chromosome interactions when meiotic recombination has to proceed in genotoxic environments. PMID:24046368

  6. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling.

    PubMed

    Yeo, Abrey J; Becherel, Olivier J; Luff, John E; Graham, Mark E; Richard, Derek; Lavin, Martin F

    2015-01-01

    Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA-DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx (-/-) pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration.

  7. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling

    PubMed Central

    Yeo, Abrey J; Becherel, Olivier J; Luff, John E; Graham, Mark E; Richard, Derek; Lavin, Martin F

    2015-01-01

    Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA–DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx−/− pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration. PMID:27462424

  8. Persistence and renaturation efficiency of thermally treated waste recombinant DNA in defined aquatic microcosms.

    PubMed

    Fu, Xiao H; Wang, Lei; Le, Yi Q; Hu, Jia J

    2012-01-01

    To validate the possibility of horizontal gene transfer (HGT) from thermally denatured recombinant DNA discharged into the eco-system, a constructed plasmid was used to investigate the persistence and renaturation efficiency of thermally denatured recombinant DNA in defined aquatic microcosms. The results revealed that there was undecayed recombinant plasmid pMDLKJ material being discharged into the aquatic microcosms even after thermal treatment at either 100°C (using boiling water) or at 120°C (using an autoclave). The plasmid had a relatively long persistence time. At least 10(2) copies μL(-1) of a specific 245 bp fragment of the plasmid could be detected after 12 h and a specific 628 bp fragment could be detected up to 2 h. The thermally denatured recombinant DNA could efficiently renature and recover its functional double stranded structure in aquatic microcosms and the highest concentration of double-stranded DNA (dsDNA) occurred around 1 h after the thermally denatured DNA was added to the system. These results imply that when thermally treated recombinant DNAs are discharged into aquatic environments, they have enough time to renature and possibly transfer to other organisms. In addition, the recombinant DNA added to aquatic microcosms could be absorbed by the seston particles in water, such as mineral, organic and colloids particles with a maximum absorption value of about 5.18 ng L(-1). This absorbed DNA could persist longer in aquatic environments than free recombinant DNA, thus further favoring HGT.

  9. Mechanism of homologous recombination and implications for aging-related deletions in mitochondrial DNA.

    PubMed

    Chen, Xin Jie

    2013-09-01

    Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells.

  10. Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

    PubMed Central

    Simon, J R; Moore, P D

    1987-01-01

    Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA. Images PMID:3302673

  11. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    SciTech Connect

    Larionov, V.; Kouprina, N. |; Edlarov, M. |; Perkins, E.; Porter, G.; Resnick, M.A.

    1993-12-31

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RADS2, RADI and the RNCI genes,

  12. Molecular Basis for Enhancement of the Meiotic DMCI Recombinase by RAD51AP1

    SciTech Connect

    Dray, Eloise; Dunlop, Myun Hwa; Kauppi, Liisa; San Filippo, Joseph San; Wiese, Claudia; Tsai, Miaw-Sheue; Begovic, Sead; Schild, David; Jasin, Maria; Keeney, Scott; Sung, Patrick

    2010-11-05

    Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 Associated Protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional co-operation is dependent on complex formation between DMC1 and RAD51AP1, and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci co-localize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.

  13. Meiotic functions of RAD18.

    PubMed

    Inagaki, Akiko; Sleddens-Linkels, Esther; Wassenaar, Evelyne; Ooms, Marja; van Cappellen, Wiggert A; Hoeijmakers, Jan H J; Seibler, Jost; Vogt, Thomas F; Shin, Myung K; Grootegoed, J Anton; Baarends, Willy M

    2011-08-15

    RAD18 is an ubiquitin ligase that is involved in replication damage bypass and DNA double-strand break (DSB) repair processes in mitotic cells. Here, we investigated the testicular phenotype of Rad18-knockdown mice to determine the function of RAD18 in meiosis, and in particular, in the repair of meiotic DSBs induced by the meiosis-specific topoisomerase-like enzyme SPO11. We found that RAD18 is recruited to a specific subfraction of persistent meiotic DSBs. In addition, RAD18 is recruited to the chromatin of the XY chromosome pair, which forms the transcriptionally silent XY body. At the XY body, RAD18 mediates the chromatin association of its interaction partners, the ubiquitin-conjugating enzymes HR6A and HR6B. Moreover, RAD18 was found to regulate the level of dimethylation of histone H3 at Lys4 and maintain meiotic sex chromosome inactivation, in a manner similar to that previously observed for HR6B. Finally, we show that RAD18 and HR6B have a role in the efficient repair of a small subset of meiotic DSBs.

  14. Male Mouse Recombination Maps for Each Autosome Identified by Chromosome Painting

    PubMed Central

    Froenicke, Lutz; Anderson, Lorinda K.; Wienberg, Johannes; Ashley, Terry

    2002-01-01

    Linkage maps constructed from genetic analysis of gene order and crossover frequency provide few clues to the basis of genomewide distribution of meiotic recombination, such as chromosome structure, that influences meiotic recombination. To bridge this gap, we have generated the first cytological recombination map that identifies individual autosomes in the male mouse. We prepared meiotic chromosome (synaptonemal complex [SC]) spreads from 110 mouse spermatocytes, identified each autosome by multicolor fluorescence in situ hybridization of chromosome-specific DNA libraries, and mapped >2,000 sites of recombination along individual autosomes, using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites. We show that SC length is strongly correlated with crossover frequency and distribution. Although the length of most SCs corresponds to that predicted from their mitotic chromosome length rank, several SCs are longer or shorter than expected, with corresponding increases and decreases in MLH1 frequency. Although all bivalents share certain general recombination features, such as few crossovers near the centromeres and a high rate of distal recombination, individual bivalents have unique patterns of crossover distribution along their length. In addition to SC length, other, as-yet-unidentified, factors influence crossover distribution leading to hot regions on individual chromosomes, with recombination frequencies as much as six times higher than average, as well as cold spots with no recombination. By reprobing the SC spreads with genetically mapped BACs, we demonstrate a robust strategy for integrating genetic linkage and physical contig maps with mitotic and meiotic chromosome structure. PMID:12432495

  15. How Good Are Indirect Tests at Detecting Recombination in Human mtDNA?

    PubMed Central

    White, Daniel James; Bryant, David; Gemmell, Neil John

    2013-01-01

    Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D′ and r2, Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ2) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7−70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed. PMID:23665874

  16. Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.

    PubMed Central

    Kitts, P A; Ayres, M D; Possee, R D

    1990-01-01

    Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location. Images PMID:2216760

  17. Molecular genetics of DNA viruses: recombinant virus technology.

    PubMed

    Neuhierl, Bernhard; Delecluse, Henri-Jacques

    2005-01-01

    Recombinant viral genomes cloned onto BAC vectors can be subjected to extensive molecular genetic analysis in the context of E. coli. Thus, the recombinant virus technology exploits the power of prokaryotic genetics to introduce all kinds of mutations into the recombinant genome. All available techniques are based on homologous recombination between a targeting vector carrying the mutated version of the gene of interest and the recombinant virus. After modification, the mutant viral genome is stably introduced into eukaryotic cells permissive for viral lytic replication. In these cells, mutant viral genomes can be packaged into infectious particles to evaluate the effect of these mutations in the context of the complete genome.

  18. No recombination of mtDNA after heteroplasmy for 50 generations in the mouse maternal germline

    PubMed Central

    Hagström, Erik; Freyer, Christoph; Battersby, Brendan J.; Stewart, James B.; Larsson, Nils-Göran

    2014-01-01

    Variants of mitochondrial DNA (mtDNA) are commonly used as markers to track human evolution because of the high sequence divergence and exclusive maternal inheritance. It is assumed that the inheritance is clonal, i.e. that mtDNA is transmitted between generations without germline recombination. In contrast to this assumption, a number of studies have reported the presence of recombinant mtDNA molecules in cell lines and animal tissues, including humans. If germline recombination of mtDNA is frequent, it would strongly impact phylogenetic and population studies by altering estimates of coalescent time and branch lengths in phylogenetic trees. Unfortunately, this whole area is controversial and the experimental approaches have been widely criticized as they often depend on polymerase chain reaction (PCR) amplification of mtDNA and/or involve studies of transformed cell lines. In this study, we used an in vivo mouse model that has had germline heteroplasmy for a defined set of mtDNA mutations for more than 50 generations. To assess recombination, we adapted and validated a method based on cloning of single mtDNA molecules in the λ phage, without prior PCR amplification, followed by subsequent mutation analysis. We screened 2922 mtDNA molecules and found no germline recombination after transmission of mtDNA under genetically and evolutionary relevant conditions in mammals. PMID:24163253

  19. A genome-wide map of mitochondrial DNA recombination in yeast.

    PubMed

    Fritsch, Emilie S; Chabbert, Christophe D; Klaus, Bernd; Steinmetz, Lars M

    2014-10-01

    In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome

  20. Effects of DNA size on transformation and recombination efficiencies in Xylella fastidiosa.

    PubMed

    Kung, Stephanie H; Retchless, Adam C; Kwan, Jessica Y; Almeida, Rodrigo P P

    2013-03-01

    Horizontally transferred DNA acquired through transformation and recombination has the potential to contribute to the diversity and evolution of naturally competent bacteria. However, many different factors affect the efficiency with which DNA can be transformed and recombined. In this study, we determined how the size of both homologous and nonhomologous regions affects transformation and recombination efficiencies in Xylella fastidiosa, a naturally competent generalist pathogen responsible for many emerging plant diseases. Our experimental data indicate that 96 bp of flanking homology is sufficient to initiate recombination, with recombination efficiencies increasing exponentially with the size of the homologous flanking region up to 1 kb. Recombination efficiencies also decreased with the size of the nonhomologous insert, with no recombination detected when 6 kb of nonhomologous DNA was flanked on either side by 1 kb of homologous sequences. Upon analyzing sequenced X. fastidiosa subsp. fastidiosa genomes for evidence of allele conversion, we estimated the mean size of recombination events to be 1,906 bp, with each event modifying, on average, 1.79% of the nucleotides in the recombined region. There is increasing evidence that horizontally acquired genes significantly affect the genetic diversity of X. fastidiosa, and DNA acquired through natural transformation could be a prominent mode of this horizontal transfer.

  1. Cyclin B-cdk activity stimulates meiotic rereplication in budding yeast.

    PubMed Central

    Strich, Randy; Mallory, Michael J; Jarnik, Michal; Cooper, Katrina F

    2004-01-01

    Haploidization of gametes during meiosis requires a single round of premeiotic DNA replication (meiS) followed by two successive nuclear divisions. This study demonstrates that ectopic activation of cyclin B/cyclin-dependent kinase in budding yeast recruits up to 30% of meiotic cells to execute one to three additional rounds of meiS. Rereplication occurs prior to the meiotic nuclear divisions, indicating that this process is different from the postmeiotic mitoses observed in other fungi. The cells with overreplicated DNA produced asci containing up to 20 spores that were viable and haploid and demonstrated Mendelian marker segregation. Genetic tests indicated that these cells executed the meiosis I reductional division and possessed a spindle checkpoint. Finally, interfering with normal synaptonemal complex formation or recombination increased the efficiency of rereplication. These studies indicate that the block to rereplication is very different in meiotic and mitotic cells and suggest a negative role for the recombination machinery in allowing rereplication. Moreover, the production of haploids, regardless of the genome content, suggests that the cell counts replication cycles, not chromosomes, in determining the number of nuclear divisions to execute. PMID:15342503

  2. Exo1 and Mre11 execute meiotic DSB end resection in the protist Tetrahymena.

    PubMed

    Lukaszewicz, Agnieszka; Shodhan, Anura; Loidl, Josef

    2015-11-01

    The resection of 5'-DNA ends at a double-strand break (DSB) is an essential step in recombinational repair, as it exposes 3' single-stranded DNA (ssDNA) tails for interaction with a repair template. In mitosis, Exo1 and Sgs1 have a conserved function in the formation of long ssDNA tails, whereas this step in the processing of programmed meiotic DSBs is less well-characterized across model organisms. In budding yeast, which has been most intensely studied in this respect, Exo1 is a major meiotic nuclease. In addition, it exerts a nuclease-independent function later in meiosis in the conversion of DNA joint molecules into ZMM-dependent crossovers. In order to gain insight into the diverse meiotic roles of Exo1, we investigated the effect of Exo1 deletion in the ciliated protist Tetrahymena. We found that Exo1 together with Mre11, but without the help of Sgs1, promotes meiotic DSB end resection. Resection is completely eliminated only if both Mre11 and Exo1 are missing. This is consistent with the yeast model where Mre11 promotes resection in the 3'-5' direction and Exo1 in the opposite 5'-3' direction. However, while the endonuclease activity of Mre11 is essential to create an entry site for exonucleases and hence to start resection in budding yeast, Tetrahymena Exo1 is able to create single-stranded DNA in the absence of Mre11. Excluding a possible contribution of the Mre11 cofactor Sae2 (Com1) as an autonomous endonuclease, we conclude that there exists another unknown nuclease that initiates DSB processing in Tetrahymena. Consistent with the absence of the ZMM crossover pathway in Tetrahymena, crossover formation is independent of Exo1.

  3. Exploration of the Dissociative Recombination following DNA ionization to DNA+ due to ionizing radiation

    NASA Astrophysics Data System (ADS)

    Strom, Richard A.; Zimmerly, Andrew T.; Andrianarijaona, Vola M.

    2014-05-01

    It is known that ionizing radiation generates low-energy secondary electrons, which may interact with the surrounding area, including biomolecules, such as triggering DNA single strand and double strand breaks as demonstrated by Sanche and coworkers (Radiat. Res. 157, 227(2002)). The bio-effects of low-energy electrons are currently a topic of high interest. Most of the studies are dedicated to dissociative electron attachments; however, the area is still mostly unexplored and still not well understood. We are computationally investigating the effect of ionizing radiation on DNA, such as its ionization to DNA+. More specifically, we are exploring the possibility of the dissociative recombination of the temporary DNA+ with one of the low-energy secondary electrons, produced by the ionizing radiation, to be another process of DNA strand breaks. Our preliminary results, which are performed with the binaries of ORCA, will be presented. Authors wish to give special thanks to Pacific Union College Student Senate in Angwin, California, for their financial support.

  4. A Maternal Screen for Genes Regulating Drosophila Oocyte Polarity Uncovers New Steps in Meiotic Progression

    PubMed Central

    Barbosa, Vitor; Kimm, Naomi; Lehmann, Ruth

    2007-01-01

    Meiotic checkpoints monitor chromosome status to ensure correct homologous recombination, genomic integrity, and chromosome segregation. In Drosophila, the persistent presence of double-strand DNA breaks (DSB) activates the ATR/Mei-41 checkpoint, delays progression through meiosis, and causes defects in DNA condensation of the oocyte nucleus, the karyosome. Checkpoint activation has also been linked to decreased levels of the TGFα-like molecule Gurken, which controls normal eggshell patterning. We used this easy-to-score eggshell phenotype in a germ-line mosaic screen in Drosophila to identify new genes affecting meiotic progression, DNA condensation, and Gurken signaling. One hundred eighteen new ventralizing mutants on the second chromosome fell into 17 complementation groups. Here we describe the analysis of 8 complementation groups, including Kinesin heavy chain, the SR protein kinase cuaba, the cohesin-related gene dPds5/cohiba, and the Tudor-domain gene montecristo. Our findings challenge the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR/Mei-41 kinase and instead reveal a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation, and Gurken protein synthesis. PMID:17507684

  5. Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family.

    PubMed

    Keeney, S; Giroux, C N; Kleckner, N

    1997-02-07

    Meiotic recombination in S. cerevisiae is initiated by double-strand breaks (DSBs). In certain mutants, breaks accumulate with a covalently attached protein, suggesting that cleavage is catalyzed by the DSB-associated protein via a topoisomerase-like transesterase mechanism. We have purified these protein-DNA complexes and identified the protein as Spo11, one of several proteins required for DSB formation. These findings strongly implicate Spo11 as the catalytic subunit of the meiotic DNA cleavage activity. This is the first identification of a biochemical function for any of the gene products involved in DSB formation. Spo11 defines a protein family with other members in fission yeast, nematodes, and archaebacteria. The S. pombe homolog, rec12p, is also known to be required for meiotic recombination. Thus, these findings provide direct evidence that the mechanism of meiotic recombination initiation is evolutionarily conserved.

  6. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects

    PubMed Central

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A.

    2016-01-01

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. PMID:27257060

  7. Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.

    PubMed

    Mbantenkhu, MacMillan; Wang, Xiaowen; Nardozzi, Jonathan D; Wilkens, Stephan; Hoffman, Elizabeth; Patel, Anamika; Cosgrove, Michael S; Chen, Xin Jie

    2011-12-09

    Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

  8. Rejuvenation of meiotic cohesion in oocytes during prophase I is required for chiasma maintenance and accurate chromosome segregation.

    PubMed

    Weng, Katherine A; Jeffreys, Charlotte A; Bickel, Sharon E

    2014-09-01

    Chromosome segregation errors in human oocytes are the leading cause of birth defects, and the risk of aneuploid pregnancy increases dramatically as women age. Accurate segregation demands that sister chromatid cohesion remain intact for decades in human oocytes, and gradual loss of the original cohesive linkages established in fetal oocytes is proposed to be a major cause of age-dependent segregation errors. Here we demonstrate that maintenance of meiotic cohesion in Drosophila oocytes during prophase I requires an active rejuvenation program, and provide mechanistic insight into the molecular events that underlie rejuvenation. Gal4/UAS inducible knockdown of the cohesion establishment factor Eco after meiotic S phase, but before oocyte maturation, causes premature loss of meiotic cohesion, resulting in destabilization of chiasmata and subsequent missegregation of recombinant homologs. Reduction of individual cohesin subunits or the cohesin loader Nipped B during prophase I leads to similar defects. These data indicate that loading of newly synthesized replacement cohesin rings by Nipped B and establishment of new cohesive linkages by the acetyltransferase Eco must occur during prophase I to maintain cohesion in oocytes. Moreover, we show that rejuvenation of meiotic cohesion does not depend on the programmed induction of meiotic double strand breaks that occurs during early prophase I, and is therefore mechanistically distinct from the DNA damage cohesion re-establishment pathway identified in G2 vegetative yeast cells. Our work provides the first evidence that new cohesive linkages are established in Drosophila oocytes after meiotic S phase, and that these are required for accurate chromosome segregation. If such a pathway also operates in human oocytes, meiotic cohesion defects may become pronounced in a woman's thirties, not because the original cohesive linkages finally give out, but because the rejuvenation program can no longer supply new cohesive linkages

  9. Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

    PubMed

    Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J

    2016-03-04

    Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity.

  10. Chromosomal context dependence of a eukaryotic recombinational hot spot.

    PubMed Central

    Ponticelli, A S; Smith, G R

    1992-01-01

    The single base-pair mutation M26 in the ade6 gene of the fission yeast Schizosaccharomyces pombe creates a hot spot for meiotic homologous recombination. When DNA fragments containing M26 and up to 3.0 kilobases of surrounding DNA were moved to the ura4 gene or to a multicopy plasmid, M26 had no detectable hot spot activity. Our results indicate that nucleotide sequences at least 1 kilobase away from M26 are required for M26 hot spot activity and suggest that, as for transcriptional promoters, a second site or proper chromatin structure is required for activation of this eukaryotic recombinational hot spot. We discuss the implications of these results for studies of other meiotic recombinational hot spots and for gene targeting. PMID:1729693

  11. 77 FR 54584 - Final Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-05

    ... resistance into a microorganism must be reviewed by the Recombinant DNA Advisory Committee (RAC) and approved... changes also clarify the criteria for determining whether an experiment to introduce drug resistance into... NIH/OBA the authority [[Page 54585

  12. How-to-Do-It: Teaching Recombinant DNA Technology in High School Biology Courses.

    ERIC Educational Resources Information Center

    Dixon, Linda

    1988-01-01

    Reports on the teaching of recombinant DNA technology in high school biology courses. Explains reactions of the public, students, and colleagues to the molecular genetics unit. Indicates equipment, curricular materials, training, workshops, and availability. (RT)

  13. DNA induces conformational changes in a recombinant human minichromosome maintenance complex.

    PubMed

    Hesketh, Emma L; Parker-Manuel, Richard P; Chaban, Yuriy; Satti, Rabab; Coverley, Dawn; Orlova, Elena V; Chong, James P J

    2015-03-20

    ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.

  14. Collaborative Learning in Biology: Debating the Ethics of Recombinant DNA Technology.

    ERIC Educational Resources Information Center

    Anderson, Rodney P.

    1998-01-01

    Discusses applications of recombinant DNA technology and the controversies surrounding that technique. Provides a cooperative learning project idea that involves teams of students investigating and debating these issues. (DDR)

  15. The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination.

    PubMed

    Burgess, Rebecca C; Rahman, Sadia; Lisby, Michael; Rothstein, Rodney; Zhao, Xiaolan

    2007-09-01

    Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8Delta mutants exhibited clonal lethality, which was due to the overamplification of 2 microm, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8Delta mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins.

  16. Alignment of Homologous Chromosomes and Effective Repair of Programmed DNA Double-Strand Breaks during Mouse Meiosis Require the Minichromosome Maintenance Domain Containing 2 (MCMDC2) Protein

    PubMed Central

    Ravindranathan, Ramya; Dereli, Ihsan; Stanzione, Marcello; Tóth, Attila

    2016-01-01

    Orderly chromosome segregation during the first meiotic division requires meiotic recombination to form crossovers between homologous chromosomes (homologues). Members of the minichromosome maintenance (MCM) helicase family have been implicated in meiotic recombination. In addition, they have roles in initiation of DNA replication, DNA mismatch repair and mitotic DNA double-strand break repair. Here, we addressed the function of MCMDC2, an atypical yet conserved MCM protein, whose function in vertebrates has not been reported. While we did not find an important role for MCMDC2 in mitotically dividing cells, our work revealed that MCMDC2 is essential for fertility in both sexes due to a crucial function in meiotic recombination. Meiotic recombination begins with the introduction of DNA double-strand breaks into the genome. DNA ends at break sites are resected. The resultant 3-prime single-stranded DNA overhangs recruit RAD51 and DMC1 recombinases that promote the invasion of homologous duplex DNAs by the resected DNA ends. Multiple strand invasions on each chromosome promote the alignment of homologous chromosomes, which is a prerequisite for inter-homologue crossover formation during meiosis. We found that although DNA ends at break sites were evidently resected, and they recruited RAD51 and DMC1 recombinases, these recombinases were ineffective in promoting alignment of homologous chromosomes in the absence of MCMDC2. Consequently, RAD51 and DMC1 foci, which are thought to mark early recombination intermediates, were abnormally persistent in Mcmdc2-/- meiocytes. Importantly, the strand invasion stabilizing MSH4 protein, which marks more advanced recombination intermediates, did not efficiently form foci in Mcmdc2-/- meiocytes. Thus, our work suggests that MCMDC2 plays an important role in either the formation, or the stabilization, of DNA strand invasion events that promote homologue alignment and provide the basis for inter-homologue crossover formation during

  17. Meiotic abnormalities in infertile males.

    PubMed

    Egozcue, J; Sarrate, Z; Codina-Pascual, M; Egozcue, S; Oliver-Bonet, M; Blanco, J; Navarro, J; Benet, J; Vidal, F

    2005-01-01

    Meiotic anomalies, as reviewed here, are synaptic chromosome abnormalities, limited to germ cells that cannot be detected through the study of the karyotype. Although the importance of synaptic errors has been underestimated for many years, their presence is related to many cases of human male infertility. Synaptic anomalies can be studied by immunostaining of synaptonemal complexes (SCs), but in this case their frequency is probably underestimated due to the phenomenon of synaptic adjustment. They can also be studied in classic meiotic preparations, which, from a clinical point of view, is still the best approach, especially if multiplex fluorescence in situ hybridization is at hand to solve difficult cases. Sperm chromosome FISH studies also provide indirect evidence of their presence. Synaptic anomalies can affect the rate of recombination of all bivalents, produce achiasmate small univalents, partially achiasmate medium-sized or large bivalents, or affect all bivalents in the cell. The frequency is variable, interindividually and intraindividually. The baseline incidence of synaptic anomalies is 6-8%, which may be increased to 17.6% in males with a severe oligozoospermia, and to 27% in normozoospermic males with one or more previous IVF failures. The clinical consequences are the production of abnormal spermatozoa that will produce a higher number of chromosomally abnormal embryos. The indications for a meiotic study in testicular biopsy are provided.

  18. Structural Studies of DNA End Detection and Resection in Homologous Recombination

    PubMed Central

    Schiller, Christian Bernd; Seifert, Florian Ulrich; Linke-Winnebeck, Christian; Hopfner, Karl-Peter

    2014-01-01

    DNA double-strand breaks are repaired by two major pathways, homologous recombination or nonhomologous end joining. The commitment to one or the other pathway proceeds via different steps of resection of the DNA ends, which is controlled and executed by a set of DNA double-strand break sensors, endo- and exonucleases, helicases, and DNA damage response factors. The molecular choreography of the underlying protein machinery is beginning to emerge. In this review, we discuss the early steps of genetic recombination and double-strand break sensing with an emphasis on structural and molecular studies. PMID:25081516

  19. Locally, meiotic double-strand breaks targeted by Gal4BD-Spo11 occur at discrete sites with a sequence preference.

    PubMed

    Murakami, Hajime; Nicolas, Alain

    2009-07-01

    Meiotic recombination is initiated by DNA double-strand breaks (DSBs) that are catalyzed by the type II topoisomerase-like Spo11 protein. Locally, at recombination hot spots, Spo11 introduces DSBs at multiple positions within approximately 75 to 250 bp, corresponding to accessible regions of the chromatin. The molecular basis of this multiplicity of cleavage positions, observed in a population of meiotic cells, remains elusive. To address this issue, we have examined the properties of the Gal4BD-Spo11 fusion protein, which targets meiotic DSBs to regions with Gal4 binding sites (UAS). By single-nucleotide resolution mapping of targeted DSBs, we found that DSB formation was restricted to discrete sites approximately 20 nucleotides from the UAS, defining a "DSB targeting window." Thus, the multiplicity of cleavage positions at natural Spo11 hot spots likely represents binding of Spo11 to different distinct sites within the accessible DNA region in each different meiotic cell. Further, we showed that mutations in the Spo11 moiety affected the DSB distribution in the DSB targeting window and that mutations in the DNA at the Spo11 cleavage site affected DSB position. These results demonstrate that Spo11 itself has sequence preference and contributes to the choice of DSB positions.

  20. Meiotic drive of chromosomal knobs reshaped the maize genome.

    PubMed Central

    Buckler, E S; Phelps-Durr, T L; Buckler, C S; Dawe, R K; Doebley, J F; Holtsford, T P

    1999-01-01

    Meiotic drive is the subversion of meiosis so that particular genes are preferentially transmitted to the progeny. Meiotic drive generally causes the preferential segregation of small regions of the genome; however, in maize we propose that meiotic drive is responsible for the evolution of large repetitive DNA arrays on all chromosomes. A maize meiotic drive locus found on an uncommon form of chromosome 10 [abnormal 10 (Ab10)] may be largely responsible for the evolution of heterochromatic chromosomal knobs, which can confer meiotic drive potential to every maize chromosome. Simulations were used to illustrate the dynamics of this meiotic drive model and suggest knobs might be deleterious in the absence of Ab10. Chromosomal knob data from maize's wild relatives (Zea mays ssp. parviglumis and mexicana) and phylogenetic comparisons demonstrated that the evolution of knob size, frequency, and chromosomal position agreed with the meiotic drive hypothesis. Knob chromosomal position was incompatible with the hypothesis that knob repetitive DNA is neutral or slightly deleterious to the genome. We also show that environmental factors and transposition may play a role in the evolution of knobs. Because knobs occur at multiple locations on all maize chromosomes, the combined effects of meiotic drive and genetic linkage may have reshaped genetic diversity throughout the maize genome in response to the presence of Ab10. Meiotic drive may be a major force of genome evolution, allowing revolutionary changes in genome structure and diversity over short evolutionary periods. PMID:10471723

  1. Stable recombination hotspots in birds.

    PubMed

    Singhal, Sonal; Leffler, Ellen M; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M; Strand, Alva I; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N; Griffith, Simon C; McVean, Gil; Przeworski, Molly

    2015-11-20

    The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but it appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking the gene that encodes PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species: the zebra finch, Taeniopygia guttata, and the long-tailed finch, Poephila acuticauda. We found that both species have recombination hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, most hotspots are shared between the two species, and their conservation seems to extend over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution.

  2. Human insulin genome sequence map, biochemical structure of insulin for recombinant DNA insulin.

    PubMed

    Chakraborty, Chiranjib; Mungantiwar, Ashish A

    2003-08-01

    Insulin is a essential molecule for type I diabetes that is marketed by very few companies. It is the first molecule, which was made by recombinant technology; but the commercialization process is very difficult. Knowledge about biochemical structure of insulin and human insulin genome sequence map is pivotal to large scale manufacturing of recombinant DNA Insulin. This paper reviews human insulin genome sequence map, the amino acid sequence of porcine insulin, crystal structure of porcine insulin, insulin monomer, aggregation surfaces of insulin, conformational variation in the insulin monomer, insulin X-ray structures for recombinant DNA technology in the synthesis of human insulin in Escherichia coli.

  3. DNA-dependent protein kinase mediates V(D)J recombination via RAG2 phosphorylation.

    PubMed

    Hah, Young-Sool; Lee, Jung Hwa; Kim, Deok Ryong

    2007-05-31

    V(D)J recombination, a site-specific gene rearrangement process occurring during the lymphocyte development, begins with DNA double strand breaks by two recombination activating gene products (RAG1/2) and finishes with the repair process by several proteins including DNA-dependent protein kinase (DNA-PK). In this report, we found that RAG2 was specifically phosphorylated by DNA-PK at the 365(th) serine residue, and this phosphorylated RAG2 affected the V(D)J recombination activity in cells in the GFP expression-based assay. While the V(D)J recombination activity between wild-type RAG2 and mutant S365A RAG2 in the assay using a signal joint substrate was undistinguishable in DNA-PK deficient cells (M059J), the activity with wild-type RAG2 was largely increased in DNA-PK proficient cells (M059K) in comparison with mutant RAG2, suggesting that RAG2 phosphorylation by DNA-PK plays a crucial role in the signal joint formation during V(D)J recombination.

  4. The impact of sequence divergence and DNA mismatch repair on homeologous recombination in Arabidopsis.

    PubMed

    Li, Liangliang; Jean, Martine; Belzile, François

    2006-03-01

    We examined the effects of substrate divergence and DNA mismatch repair (MMR) on recombination in Arabidopsis thaliana. Relative to the frequency observed in plants with a homologous construct (0% divergence), recombination was decreased 4.1-, 9.6-, 11.7- or 20.3-fold, respectively, in lines with constructs containing 0.5%, 2%, 4% or 9% divergence between the recombination substrates. To evaluate the contribution of the MMR system in this decrease, 12 independent reporter lines (two or three lines per reporter construct) were crossed to an AtMSH2 T-DNA insertional mutant. We examined the recombination frequency in progeny homozygous for a reporter T-DNA and homozygous either for the wild type or the mutant allele of AtMSH2. The loss of MMR activity led to a two- to ninefold increase in homeologous recombination and the size of the increase did not seem to correlate with the amount of divergence. Inversely, complementation of the insertional mutant with a wild-type cDNA of AtMSH2 reduced recombination. Our results demonstrate clearly that sequence divergence can dramatically reduce the recombination frequency in plants and that the MMR system plays a part in this decrease.

  5. Budding Yeast SLX4 Contributes to the Appropriate Distribution of Crossovers and Meiotic Double-Strand Break Formation on Bivalents During Meiosis

    PubMed Central

    Higashide, Mika; Shinohara, Miki

    2016-01-01

    The number and distribution of meiosis crossover (CO) events on each bivalent are strictly controlled by multiple mechanisms to assure proper chromosome segregation during the first meiotic division. In Saccharomyces cerevisiae, Slx4 is a multi-functional scaffold protein for structure-selective endonucleases, such as Slx1 and Rad1 (which are involved in DNA damage repair), and is also a negative regulator of the Rad9-dependent signaling pathway with Rtt107. Slx4 has been believed to play only a minor role in meiotic recombination. Here, we report that Slx4 is involved in proper intrachromosomal distribution of meiotic CO formation, especially in regions near centromeres. We observed an increase in uncontrolled CO formation only in a region near the centromere in the slx4∆ mutant. Interestingly, this phenomenon was not observed in the slx1∆, rad1∆, or rtt107∆ mutants. In addition, we observed a reduced number of DNA double-strand breaks (DSBs) and altered meiotic DSB distribution on chromosomes in the slx4∆ mutant. This suggests that the multi-functional Slx4 is required for proper CO formation and meiotic DSB formation. PMID:27172214

  6. Quantification and persistence of recombinant DNA of Roundup Ready corn and soybean in rotation.

    PubMed

    Lerat, Sylvain; Gulden, Robert H; Hart, Miranda M; Powell, Jeff R; England, Laura S; Pauls, K Peter; Swanton, Clarence J; Klironomos, John N; Trevors, Jack T

    2007-12-12

    The presence of the recombinant cp4 epsps gene from Roundup Ready (RR) corn and RR soybean was quantified using real-time PCR in soil samples from a field experiment growing RR and conventional corn and soybean in rotation. RR corn and RR soybean cp4 epsps persisted in soil for up to 1 year after seeding. The concentration of recombinant DNA in soil peaked in July and August in RR corn and RR soybean plots, respectively. A small fraction of soil samples from plots seeded with conventional crops contained recombinant DNA, suggesting transgene dispersal by means of natural process or agricultural practices. This research will aid in the understanding of the persistence of recombinant DNA in agricultural cropping systems.

  7. Biparental inheritance of organelles in Pelargonium: evidence for intergenomic recombination of mitochondrial DNA.

    PubMed

    Apitz, Janina; Weihe, Andreas; Pohlheim, Frank; Börner, Thomas

    2013-02-01

    While uniparental transmission of mtDNA is widespread and dominating in eukaryotes leaving mutation as the major source of genotypic diversity, recently, biparental inheritance of mitochondrial genes has been demonstrated in reciprocal crosses of Pelargonium zonale and P. inquinans. The thereby arising heteroplasmy carries the potential for recombination between mtDNAs of different descent, i.e. between the parental mitochondrial genomes. We have analyzed these Pelargonium hybrids for mitochondrial intergenomic recombination events by examining differences in DNA blot hybridization patterns of the mitochondrial genes atp1 and cob. Further investigation of these genes and their flanking regions using nucleotide sequence polymorphisms and PCR revealed DNA segments in the progeny, which contained both P. zonale and P. inquinans sequences suggesting an intergenomic recombination in hybrids of Pelargonium. This turns Pelargonium into an interesting subject for studies of recombination and evolutionary dynamics of mitochondrial genomes.

  8. Self-regulation of recombinant DNA technology in Japan in the 1970s.

    PubMed

    Nagai, Hiroyuki; Nukaga, Yoshio; Saeki, Koji; Akabayashi, Akira

    2009-07-01

    Recombinant DNA technology was developed in the United States in the early 1970s. Leading scientists held an international Asilomar Conference in 1975 to examine the self regulation of recombinant DNA technology, followed by the U.S. National Institutes of Health drafting the Recombinant DNA Research Guidelines in 1976. The result of this conference significantly affected many nations, including Japan. However, there have been few historical studies on the self-regulation of recombinant technologies conducted by scientists and government officials in Japan. The purpose of this paper is to analyze how the Science Council of Japan, the Ministry of Education, Science adn Culture, and the Science and Technology Agency developed self-regulation policies for recombinant DNA technology in Japan in the 1970s. Groups of molecular biologist and geneticists played a key role in establishing guidelines in cooperation with government officials. Our findings suggest that self-regulation policies on recombinant DNA technology have influenced safety management for the life sciences and establishment of institutions for review in Japan.

  9. Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair

    SciTech Connect

    Marchetti, Francesco; Bishop, Jack; Gingerich, John; Wyrobek, Andrew J.

    2015-01-08

    De novo point mutations and chromosomal structural aberrations (CSA) detected in offspring of unaffected parents show a preferential paternal origin with higher risk for older fathers. Studies in rodents suggest that heritable mutations transmitted from the father can arise from either paternal or maternal misrepair of damaged paternal DNA, and that the entire spermatogenic cycle can be at risk after mutagenic exposure. Understanding the susceptibility and mechanisms of transmission of paternal mutations is important in family planning after chemotherapy and donor selection for assisted reproduction. We report that treatment of male mice with melphalan (MLP), a bifunctional alkylating agent widely used in chemotherapy, induces DNA lesions during male mouse meiosis that persist unrepaired as germ cells progress through DNA repair-competent phases of spermatogenic development. After fertilization, unrepaired sperm DNA lesions are mis-repaired into CSA by the egg's DNA repair machinery producing chromosomally abnormal offspring. In conclusion, these findings highlight the importance of both pre- and post-fertilization DNA repair in assuring the genomic integrity of the conceptus.

  10. Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair

    DOE PAGES

    Marchetti, Francesco; Bishop, Jack; Gingerich, John; ...

    2015-01-08

    De novo point mutations and chromosomal structural aberrations (CSA) detected in offspring of unaffected parents show a preferential paternal origin with higher risk for older fathers. Studies in rodents suggest that heritable mutations transmitted from the father can arise from either paternal or maternal misrepair of damaged paternal DNA, and that the entire spermatogenic cycle can be at risk after mutagenic exposure. Understanding the susceptibility and mechanisms of transmission of paternal mutations is important in family planning after chemotherapy and donor selection for assisted reproduction. We report that treatment of male mice with melphalan (MLP), a bifunctional alkylating agent widelymore » used in chemotherapy, induces DNA lesions during male mouse meiosis that persist unrepaired as germ cells progress through DNA repair-competent phases of spermatogenic development. After fertilization, unrepaired sperm DNA lesions are mis-repaired into CSA by the egg's DNA repair machinery producing chromosomally abnormal offspring. In conclusion, these findings highlight the importance of both pre- and post-fertilization DNA repair in assuring the genomic integrity of the conceptus.« less

  11. Divergent kleisin subunits of cohesin specify mechanisms to tether and release meiotic chromosomes.

    PubMed

    Severson, Aaron F; Meyer, Barbara J

    2014-08-29

    We show that multiple, functionally specialized cohesin complexes mediate the establishment and two-step release of sister chromatid cohesion that underlies the production of haploid gametes. In C. elegans, the kleisin subunits REC-8 and COH-3/4 differ between meiotic cohesins and endow them with distinctive properties that specify how cohesins load onto chromosomes and then trigger and release cohesion. Unlike REC-8 cohesin, COH-3/4 cohesin becomes cohesive through a replication-independent mechanism initiated by the DNA double-stranded breaks that induce crossover recombination. Thus, break-induced cohesion also tethers replicated meiotic chromosomes. Later, recombination stimulates separase-independent removal of REC-8 and COH-3/4 cohesins from reciprocal chromosomal territories flanking the crossover site. This region-specific removal likely underlies the two-step separation of homologs and sisters. Unexpectedly, COH-3/4 performs cohesion-independent functions in synaptonemal complex assembly. This new model for cohesin function diverges from that established in yeast but likely applies directly to plants and mammals, which utilize similar meiotic kleisins.

  12. Saccharomyces cerevisiae Dmc1 and Rad51 proteins preferentially function with Tid1 and Rad54 proteins, respectively, to promote DNA strand invasion during genetic recombination.

    PubMed

    Nimonkar, Amitabh V; Dombrowski, Christopher C; Siino, Joseph S; Stasiak, Alicja Z; Stasiak, Andrzej; Kowalczykowski, Stephen C

    2012-08-17

    The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

  13. Replication-dependent early meiotic requirement for Spo11 and Rad50.

    PubMed

    Merino, S T; Cummings, W J; Acharya, S N; Zolan, M E

    2000-09-12

    Spo11 and the Rad50-Mre11 complex have been indirectly implicated in processes associated with DNA replication. These proteins also have been shown to have early meiotic roles essential for the formation of a programmed DNA double-strand break known in Saccharomyces cerevisiae to initiate meiotic recombination. In both S. cerevisiae and the basidiomycete Coprinus cinereus, spo11 and rad50 mutants are defective in chromosome synapsis during meiosis. Here we demonstrate that a partial restoration of synapsis occurs in C. cinereus spo11 and rad50 mutants if premeiotic DNA replication is prevented. Double mutants were constructed with spo11-1 or rad50-4 and another mutant, spo22-1, which does not undergo premeiotic DNA replication. In both cases, we observed an increase in the percentage of nuclei containing synaptonemal complex (SC) structures, with concomitant decreases in the percentage of nuclei containing axial elements (AE) only or no structures. Both types of double mutants demonstrated significant increases in the average numbers of AE and SC, although SC-containing nuclei did not on average contain more AE than did nuclei showing no synapsis. Our results show that Spo11-induced recombination is not absolutely required for synapsis in C. cinereus, and that the early meiotic role of both Spo11 and Rad50 in SC formation partially depends on premeiotic S phase. This dependency likely reflects either a requirement for these proteins imposed by the premeiotic replication process itself or a requirement for these proteins in synapsis when a sister chromatid (the outcome of DNA replication) is present.

  14. Waste recombinant DNA: effectiveness of thermo-treatment to manage potential gene pollution.

    PubMed

    Fu, Xiaohua; Li, Mengnan; Zheng, Guanghong; Le, Yiquan; Wang, Lei

    2009-01-01

    Heating at 100 degrees C for 5-10 min is a common method for treating wastewater containing recombinant DNA in many bio-laboratories in China. In this experiment, plasmid pET-28b was used to investigate decay efficiency of waste recombinant DNA during thermo-treatment. The results showed that the decay half-life of the plasmid was 2.7-4.0 min during the thermo-treatment, and even heating for 30 min the plasmids still retained some transforming activity. Low pH promoted the decay of recombinant DNA, but NaCl, bovine serum albumin and EDTA, which existed in the most wastewater from bio-laboratories, protected DNA from degradation. Thus, the decay half-life of plasmid DNA may be longer than 2.7-4.0 min practically. These results suggest that the effectiveness of heating at 100 degrees C for treating waste recombinant DNA is low and a gene pollution risk remains when those thermo-treated recombinant DNAs are discharged into the environment. Therefore other simple and effective methods should be developed.

  15. Recombinant DNA Paper Model Simulation: The Genetic Engineer.

    ERIC Educational Resources Information Center

    Wagner, Joan

    1998-01-01

    Describes a course for talented high school students that focuses on DNA science and technology. Employs Cold Spring Harbor's DNA Science laboratory manual. Engages students in performing sickle-cell anemia and thalassemia tests in rabbits. (DDR)

  16. Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli.

    PubMed Central

    Moreau, P L

    1988-01-01

    Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA. PMID:2836358

  17. Switch recombination breakpoints are strictly correlated with DNA recognition motifs for immunoglobulin S gamma 3 DNA-binding proteins

    PubMed Central

    1992-01-01

    The deletion looping out model of switch (S) recombination predicts that the intervening DNA between switch regions will be excised as a circle. Circular excision products of immunoglobulin switch recombination have been recently isolated from lipopolysaccharide (LPS)- stimulated spleen cells. The recombination breakpoints in these large circles were found to fall within switch regions. Since switch recombination is clearly focused on switch regions, we hypothesized that some DNA-binding protein factor might be involved in specifically recognizing and facilitating the alignment of switch regions before recombination. Two DNA-binding proteins that specifically interact with two discrete regions of the S gamma 3 tandem repeat have been identified in crude and partially purified nuclear extracts derived from LPS- and dextran sulfate (DxS)-activated splenic B cells. The first factor has been found indistinguishable from NF-kappa B by mobility shift assays, methylation interference, competition binding studies, and supershift analysis using an antiserum specific for the p50 component. The second appears to be composed of two closely traveling mobilities that do not separate upon partial purification. This second complex is unique and specific for S gamma 3 by methylation interference assays and competition-binding analysis. The sites at which recombination occurs in the S gamma 3 switch region have been analyzed and found to strictly correlate with the binding sites of the S gamma 3 switch binding proteins. PMID:1500850

  18. Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration.

    PubMed

    Wilson, Marenda A; Kwon, YoungHo; Xu, Yuanyuan; Chung, Woo-Hyun; Chi, Peter; Niu, Hengyao; Mayle, Ryan; Chen, Xuefeng; Malkova, Anna; Sung, Patrick; Ira, Grzegorz

    2013-10-17

    During DNA repair by homologous recombination (HR), DNA synthesis copies information from a template DNA molecule. Multiple DNA polymerases have been implicated in repair-specific DNA synthesis, but it has remained unclear whether a DNA helicase is involved in this reaction. A good candidate DNA helicase is Pif1, an evolutionarily conserved helicase in Saccharomyces cerevisiae important for break-induced replication (BIR) as well as HR-dependent telomere maintenance in the absence of telomerase found in 10-15% of all cancers. Pif1 has a role in DNA synthesis across hard-to-replicate sites and in lagging-strand synthesis with polymerase δ (Polδ). Here we provide evidence that Pif1 stimulates DNA synthesis during BIR and crossover recombination. The initial steps of BIR occur normally in Pif1-deficient cells, but Polδ recruitment and DNA synthesis are decreased, resulting in premature resolution of DNA intermediates into half-crossovers. Purified Pif1 protein strongly stimulates Polδ-mediated DNA synthesis from a D-loop made by the Rad51 recombinase. Notably, Pif1 liberates the newly synthesized strand to prevent the accumulation of topological constraint and to facilitate extensive DNA synthesis via the establishment of a migrating D-loop structure. Our results uncover a novel function of Pif1 and provide insights into the mechanism of HR.

  19. Preparations of Meiotic Pachytene Chromosomes and Extended DNA Fibers from Cotton Suitable for Fluorescence In Situ Hybridization

    PubMed Central

    Liu, Fang; Ling, Jian; Wang, Chunying; Li, Shaohui; Zhang, Xiangdi; Wang, Yuhong; Wang, Kunbo

    2012-01-01

    Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established

  20. A simplified method for purification of recombinant soluble DnaA proteins.

    PubMed

    Zawilak-Pawlik, Anna M; Kois, Agnieszka; Zakrzewska-Czerwinska, Jolanta

    2006-07-01

    An improved, simplified method for the purification of recombinant, tagged DnaA proteins is described. The presented protocol allowed us to purify soluble DnaA proteins from two different bacterial species: Helicobacter pylori and Streptomyces coelicolor, but it can most likely also be used for the isolation of DnaA proteins from other bacteria, as it was adapted for Mycobacterium tuberculosis DnaA. The isolation procedure consists of protein precipitation with ammonium sulphate followed by affinity chromatography. The composition of the buffers used at each purification step is crucial for the successful isolation of the recombinant DnaA proteins. The universality of the method in terms of its application to differently tagged proteins (His-tagged or GST-tagged) as well as different properties of purified proteins (e.g., highly aggregating truncated forms) makes the protocol highly useful for all studies requiring purified and active DnaA proteins.

  1. A simplified mathematical model of directional DNA site-specific recombination by serine integrases

    PubMed Central

    Zhao, Jia; Stark, W. Marshall; Colloms, Sean D.; Ebenhöh, Oliver

    2017-01-01

    Serine integrases catalyse site-specific recombination to integrate and excise bacteriophage genomes into and out of their host's genome. These enzymes exhibit remarkable directionality; in the presence of the integrase alone, recombination between attP and attB DNA sites is efficient and irreversible, giving attL and attR products which do not recombine further. However, in the presence of the bacteriophage-encoded recombination directionality factor (RDF), integrase efficiently promotes recombination between attL and attR to re-form attP and attB. The DNA substrates and products of both reactions are approximately isoenergetic, and no cofactors (such as adenosine triphosphate) are required for recombination. The thermodynamic driving force for directionality of these reactions is thus enigmatic. Here, we present a minimal mathematical model which can explain the directionality and regulation of both ‘forward’ and ‘reverse’ reactions. In this model, the substrates of the ‘forbidden’ reactions (between attL and attR in the absence of RDF, attP and attB in the presence of RDF) are trapped as inactive protein–DNA complexes, ensuring that these ‘forbidden’ reactions are extremely slow. The model is in good agreement with the observed in vitro kinetics of recombination by ϕC31 integrase, and defines core features of the system necessary and sufficient for directionality. PMID:28077763

  2. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    PubMed Central

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

    2014-01-01

    Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

  3. Mismatch repair proteins: key regulators of genetic recombination.

    PubMed

    Surtees, J A; Argueso, J L; Alani, E

    2004-01-01

    Mismatch repair (MMR) systems are central to maintaining genome stability in prokaryotes and eukaryotes. MMR proteins play a fundamental role in avoiding mutations, primarily by removing misincorporation errors that occur during DNA replication. MMR proteins also act during genetic recombination in steps that include repairing mismatches in heteroduplex DNA, modulating meiotic crossover control, removing 3' non-homologous tails during double-strand break repair, and preventing recombination between divergent sequences. In this review we will, first, discuss roles for MMR proteins in repairing mismatches that occur during recombination, particularly during meiosis. We will also explore how studying this process has helped to refine models of double-strand break repair, and particularly to our understanding of gene conversion gradients. Second, we will examine the role of MMR proteins in repressing homeologous recombination, i.e. recombination between divergent sequences. We will also compare the requirements for MMR proteins in preventing homeologous recombination to the requirements for these proteins in mismatch repair.

  4. The evolution of meiotic sex and its alternatives

    PubMed Central

    Mirzaghaderi, Ghader

    2016-01-01

    Meiosis is an ancestral, highly conserved process in eukaryotic life cycles, and for all eukaryotes the shared component of sexual reproduction. The benefits and functions of meiosis, however, are still under discussion, especially considering the costs of meiotic sex. To get a novel view on this old problem, we filter out the most conserved elements of meiosis itself by reviewing the various modifications and alterations of modes of reproduction. Our rationale is that the indispensable steps of meiosis for viability of offspring would be maintained by strong selection, while dispensable steps would be variable. We review evolutionary origin and processes in normal meiosis, restitutional meiosis, polyploidization and the alterations of meiosis in forms of uniparental reproduction (apomixis, apomictic parthenogenesis, automixis, selfing) with a focus on plants and animals. This overview suggests that homologue pairing, double-strand break formation and homologous recombinational repair at prophase I are the least dispensable elements, and they are more likely optimized for repair of oxidative DNA damage rather than for recombination. Segregation, ploidy reduction and also a biparental genome contribution can be skipped for many generations. The evidence supports the theory that the primary function of meiosis is DNA restoration rather than recombination. PMID:27605505

  5. PRDM9 sticks its zinc fingers into recombination hotspots and between species.

    PubMed

    Sandovici, Ionel; Sapienza, Carmen

    2010-05-24

    Meiotic recombination events typically cluster within narrow regions of the genome termed hotspots. A series of recent papers reveals that PRDM9, a C2H2-type zinc-finger protein with histone H3 lysine 4 methyltransferase activity, plays a major role in the specification of hotspots. The zinc fingers that contact DNA in a sequence-dependent manner evolve rapidly and are under positive selection, leading to differences in the location of recombination hotspots as well as hybrid sterility.

  6. Rad51 and Rad52 Are Involved in Homologous Recombination of Replicating Herpes Simplex Virus DNA

    PubMed Central

    Tang, Ka-Wei; Norberg, Peter; Holmudden, Martin; Elias, Per; Liljeqvist, Jan-Åke

    2014-01-01

    Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells. PMID:25365323

  7. Rad51 and Rad52 are involved in homologous recombination of replicating herpes simplex virus DNA.

    PubMed

    Tang, Ka-Wei; Norberg, Peter; Holmudden, Martin; Elias, Per; Liljeqvist, Jan-Åke

    2014-01-01

    Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells.

  8. Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli

    PubMed Central

    Thomason, Lynn C.; Costantino, Nina

    2016-01-01

    ABSTRACT Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. PMID:27624131

  9. A facile method for reversibly linking a recombinant protein to DNA.

    PubMed

    Goodman, Russell P; Erben, Christoph M; Malo, Jonathan; Ho, Wei M; McKee, Mireya L; Kapanidis, Achillefs N; Turberfield, Andrew J

    2009-06-15

    We present a facile method for linking recombinant proteins to DNA. It is based on the nickel-mediated interaction between a hexahistidine tag (His(6)-tag) and DNA functionalized with three nitrilotriacetic acid (NTA) groups. The resulting DNA-protein linkage is site-specific. It can be broken quickly and controllably by the addition of a chelating agent that binds nickel. We have used this new linker to bind proteins to a variety of DNA motifs commonly used in the fabrication of nanostructures by DNA self-assembly.

  10. The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery.

    PubMed

    Kidane, Dawit; Ayora, Silvia; Sweasy, Joann B; Graumann, Peter L; Alonso, Juan C

    2012-01-01

    Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

  11. The Landscape of Mouse Meiotic Double-Strand Break Formation, Processing, and Repair.

    PubMed

    Lange, Julian; Yamada, Shintaro; Tischfield, Sam E; Pan, Jing; Kim, Seoyoung; Zhu, Xuan; Socci, Nicholas D; Jasin, Maria; Keeney, Scott

    2016-10-20

    Heritability and genome stability are shaped by meiotic recombination, which is initiated via hundreds of DNA double-strand breaks (DSBs). The distribution of DSBs throughout the genome is not random, but mechanisms molding this landscape remain poorly understood. Here, we exploit genome-wide maps of mouse DSBs at unprecedented nucleotide resolution to uncover previously invisible spatial features of recombination. At fine scale, we reveal a stereotyped hotspot structure-DSBs occur within narrow zones between methylated nucleosomes-and identify relationships between SPO11, chromatin, and the histone methyltransferase PRDM9. At large scale, DSB formation is suppressed on non-homologous portions of the sex chromosomes via the DSB-responsive kinase ATM, which also shapes the autosomal DSB landscape at multiple size scales. We also provide a genome-wide analysis of exonucleolytic DSB resection lengths and elucidate spatial relationships between DSBs and recombination products. Our results paint a comprehensive picture of features governing successive steps in mammalian meiotic recombination.

  12. Engineering cellulosic bioreactors by template assisted DNA shuffling and in vitro recombination (TADSir).

    PubMed

    Davis, Leroy K

    2014-10-01

    The current study focuses on development of a bioreactor engineering strategy based on exploitation of the Arabidopsis thaliana genome. Chimeric A. thaliana glycosyl hydrolase (GH) gene libraries were assembled using a novel directed evolution strategy (TADSir: template assisted DNA shuffling and in vitro recombination) that promotes DNA recombination by reassembly of DNA fragments on unique gene templates. TADSir was modeled using a set of algorithms designed to simulate DNA interactions based on nearest neighbor base stacking interactions and Gibb's free energy differences between helical coil and folded DNA states. The algorithms allow for target gene prediction and for in silica analysis of chimeric gene library composition. Further, the study investigated utilization of A. thaliana GH sequence space for bioreactor design by evolving 20 A. thaliana genes representing the GH1, GH3, GH5, GH9 and GH10 gene families. Notably, TADSir achieved streamlined engineering of Saccharomyces cerevisiae and spinach mesophyll protoplast bioreactors capable of processing CM cellulose, Avicel and xylan.

  13. V(D)J recombination coding junction formation without DNA homology: processing of coding termini.

    PubMed Central

    Boubnov, N V; Wills, Z P; Weaver, D T

    1993-01-01

    Coding junction formation in V(D)J recombination generates diversity in the antigen recognition structures of immunoglobulin and T-cell receptor molecules by combining processes of deletion of terminal coding sequences and addition of nucleotides prior to joining. We have examined the role of coding end DNA composition in junction formation with plasmid substrates containing defined homopolymers flanking the recombination signal sequence elements. We found that coding junctions formed efficiently with or without terminal DNA homology. The extent of junctional deletion was conserved independent of coding ends with increased, partial, or no DNA homology. Interestingly, G/C homopolymer coding ends showed reduced deletion regardless of DNA homology. Therefore, DNA homology cannot be the primary determinant that stabilizes coding end structures for processing and joining. PMID:8413286

  14. DNA sequence and structure requirements for cleavage of V(D)J recombination signal sequences.

    PubMed Central

    Cuomo, C A; Mundy, C L; Oettinger, M A

    1996-01-01

    Purified RAG1 and RAG2 proteins can cleave DNA at V(D)J recombination signals. In dissecting the DNA sequence and structural requirements for cleavage, we find that the heptamer and nonamer motifs of the recombination signal sequence can independently direct both steps of the cleavage reaction. Proper helical spacing between these two elements greatly enhances the efficiency of cleavage, whereas improper spacing can lead to interference between the two elements. The signal sequences are surprisingly tolerant of structural variation and function efficiently when nicks, gaps, and mismatched bases are introduced or even when the signal sequence is completely single stranded. Sequence alterations that facilitate unpairing of the bases at the signal/coding border activate the cleavage reaction, suggesting that DNA distortion is critical for V(D)J recombination. PMID:8816481

  15. Involvement of DNA-dependent RNA polymerase in a recA-independent pathway of genetic recombination in Escheria coli.

    PubMed

    Ikeda, H; Kobayashi, I

    1977-09-01

    Recombinant DNA molecule of phage lambda formed in Escherichia coli in the presence of chloramphenicol and/or rifampin can be assayed by their biological activity. recA- cells were found to be capable of forming recombinant lambda phage DNA in the presence of chloramphenicol. The relatively high recA-independent recombination observed in this system contrasts with the relatively low recA-independent recombination when recombinant phage particles rather than recombinant DNA are titrated. Formation of the recombinant DNA was suppressed by the the addition of rifampin. The introduction of the rif-r mutation into host bacteria made their recombination activity rifampin-resistant. These results show that DNA-dependent RNA polymerase (EC 2.7.7.6) is involved in this recA-independent pathway of recombination, which is named the "Rpo pathway." This is distinct from Red, Int, RecBC, RecE, or Der pathways of recombination. Crossover was much more frequent in the N-PL-cI and cI-PR-O regions than in the A-D and O-S regions. The crossover seems to occur in the regions that are transcribed actively. Some local change of DNA structure caused by transcription might be required for the Rpo pathway of recombination.

  16. An inhibitor of DNA recombination blocks memory consolidation, but not reconsolidation, in context fear conditioning.

    PubMed

    Colón-Cesario, Melissa; Wang, Jianpeng; Ramos, Xiomara; García, Hermes G; Dávila, Jorge J; Laguna, Jessenia; Rosado, Claribel; Peña de Ortiz, Sandra

    2006-05-17

    Genomic recombination requires cutting, processing, and rejoining of DNA by endonucleases, polymerases, and ligases, among other factors. We have proposed that DNA recombination mechanisms may contribute to long-term memory (LTM) formation in the brain. Our previous studies with the nucleoside analog 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP), a known inhibitor of DNA ligases and polymerases, showed that this agent blocked consolidation of conditioned taste aversion without interfering with short-term memory (STM). However, because polymerases and ligases are also essential for DNA replication, it remained unclear whether the effects of this drug on consolidation were attributable to interference with DNA recombination or neurogenesis. Here we show, using C57BL/6 mice, that ara-CTP specifically blocks consolidation but not STM of context fear conditioning, a task previously shown not to require neurogenesis. The effects of a single systemic dose of cytosine arabinoside (ara-C) on LTM were evident as early as 6 h after training. In addition, although ara-C impaired LTM, it did not impair general locomotor activity nor induce brain neurotoxicity. Importantly, hippocampal, but not insular cortex, infusions of ara-C also blocked consolidation of context fear conditioning. Separate studies revealed that context fear conditioning training significantly induced nonhomologous DNA end joining activity indicative of DNA ligase-dependent recombination in hippocampal, but not cortex, protein extracts. Finally, unlike inhibition of protein synthesis, systemic ara-C did not block reconsolidation of context fear conditioning. Our results support the idea that DNA recombination is a process specific to consolidation that is not involved in the postreactivation editing of memories.

  17. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    SciTech Connect

    Larionov, V.; Kouprina, N. |; Eldarov, M. |; Perkins, E.; Porter, G.; Resnick, M.A.

    1994-10-01

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic-growth. The frequency of recombination is partly dependent on the method of transformation In that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RAD52, RAD1 and the RNC1 genes.

  18. High-frequency intermolecular homologous recombination during herpes simplex virus-mediated plasmid DNA replication.

    PubMed

    Fu, Xinping; Wang, Hua; Zhang, Xiaoliu

    2002-06-01

    Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.

  19. Recombination between DNA repeats in yeast hpr1delta cells is linked to transcription elongation.

    PubMed Central

    Prado, F; Piruat, J I; Aguilera, A

    1997-01-01

    The induction of recombination by transcription activation has been documented in prokaryotes and eukaryotes. Unwinding of the DNA duplex, disruption of chromatin structure or changes in local supercoiling associated with transcription can be indirectly responsible for the stimulation of recombination. Here we provide genetic and molecular evidence for a specific mechanism of stimulation of recombination by transcription. We show that the induction of deletions between repeats in hpr1delta cells of Saccharomyces cerevisiae is linked to transcription elongation. Molecular analysis of different direct repeat constructs reveals that deletions induced by hpr1delta are specific for repeat constructs in which transcription initiating at an external promoter traverses particular regions of the DNA flanked by the repeats. Transcription becomes HPR1 dependent when elongating through such regions. Both the induction of deletions and the HPR1 dependence of transcription were abolished when a strong terminator was used to prevent transcription from proceeding through the DNA region flanked by the repeats. In contrast to previously reported cases of transcription-induced recombination, there was no correlation between high levels of transcripts and high levels of recombination. Our study provides evidence that direct repeat recombination can be induced by transcriptional elongation. PMID:9184227

  20. Control of helicase loading in the coupled DNA replication and recombination systems of bacteriophage T4.

    PubMed

    Branagan, Amy M; Klein, Jenny A; Jordan, Christian S; Morrical, Scott W

    2014-01-31

    The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates.

  1. Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

    PubMed Central

    Bebel, Aleksandra; Karaca, Ezgi; Kumar, Banushree; Stark, W Marshall; Barabas, Orsolya

    2016-01-01

    Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division. DOI: http://dx.doi.org/10.7554/eLife.19706.001 PMID:28009253

  2. Immunogenicity analysis following human immunodeficiency virus recombinant DNA and recombinant vaccinia virus Tian Tan prime-boost immunization.

    PubMed

    Liu, Cunxia; Du, Shouwen; Li, Chang; Wang, Yuhang; Wang, Maopeng; Li, Yi; Yin, Ronglan; Li, Xiao; Ren, Dayong; Qin, Yanqing; Ren, Jingqiang; Jin, Ningyi

    2013-06-01

    This study assessed and compared the immunogenicity of various immunization strategies in mice using combinations of recombinant DNA (pCCMp24) and recombinant attenuated vaccinia virus Tian Tan (rddVTT-CCMp24). Intramuscular immunization was performed on days 0 (prime) and 21 (boost). The immunogenicity of the vaccine schedules was determined by measuring human immunodeficiency virus (HIV)-specific binding antibody levels and cytokine (interleukin-2 and interleukin-4) concentrations in peripheral blood, analyzing lymphocyte proliferation capacity against HIV epitopes and CD4(+)/CD8(+) cell ratio, and monitoring interferon-gamma levels at different times post-immunization. The results showed that pCCMp24, rddVTT-CCMp24 and their prime-boost immunization induced humoral and cellular immune responses. The pCCMp24/rddVTT-CCMp24 immunization strategy increased CD8(+) T cells and induced more IFN-γ-secreting cells compared with single-shot rDNA. The prime-boost immunization strategy also induced the generation of cellular immunological memory to HIV epitope peptides. These results demonstrated that prime-boost immunization with rDNA and rddVTT-CCMp24 had a tendency to induce greater cellular immune response than single-shot vaccinations, especially IFN-γ response, providing a basis for further studies.

  3. The OECD Blue Book on Recombinant DNA Safety Considerations: it's influence on ISBR and EFSA activities.

    PubMed

    Schiemann, Joachim

    2006-01-01

    Biosafety regulatory frameworks are intended to serve as mechanisms for ensuring the safe use of biotechnology products without imposing unacceptable risk to human health or the environment, or unintended constraints to technology transfer. The OECD Blue Book on "Recombinant DNA Safety Considerations", setting out principles and concepts for handling genetically modified organisms safely outside of contained laboratory conditions, was a milestone in the history of biotechnology. The "Recombinant DNA Safety Considerations" definitively became the major resource for the formulation of national regulatory frameworks and international regulations, including the Cartagena Protocol.

  4. Spo11 and the Formation of DNA Double-Strand Breaks in Meiosis.

    PubMed

    Keeney, Scott

    2008-01-01

    Meiotic recombination is carried out through a specialized pathway for the formation and repair of DNA double-strand breaks made by the Spo11 protein, a relative of archaeal topoisomerase VI. This review summarizes recent studies that provide insight to the mechanism of DNA cleavage by Spo11, functional interactions of Spo11 with other proteins required for break formation, mechanisms that control the timing of recombination initiation, and evolutionary conservation and divergence of these processes.

  5. [Important points in virus research using recombinant DNA technology].

    PubMed

    Nikaido, Takahiko; Takeuchi, Kaoru

    2007-06-01

    Cartagena Protocol on Biosafety to the Convention on Biological Diversity seeks to protect biological diversity from potential risks posed by living modified organisms (LMOs) resulting from modern biotechnology. This protocol was ratified in Japan after establishing domestic law and regulations for the protocol. In the domestic law, use of LMOs is classified into type 1 use (use without containment measures) and type 2 use (use with containment measures). According to the domestic law, most of experiments using recombinant viruses are required for the approval of the Minister. In this article, we will explain Cartagena Protocol and the Japanese domestic low and indicate an example of application form for the approval of the Minister.

  6. Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status.

    PubMed

    Ismail, Ruzila; Allaudin, Zeenathul Nazariah; Lila, Mohd-Azmi Mohd

    2012-09-07

    Gene therapy and vaccines are rapidly developing field in which recombinant nucleic acids are introduced in mammalian cells for enhancement, restoration, initiation or silencing biochemical function. Beside simplicity in manipulation and rapid manufacture process, plasmid DNA-based vaccines have inherent features that make them promising vaccine candidates in a variety of diseases. This present review focuses on the safety concern of the genetic elements of plasmid such as propagation and expression units as well as their host genome for the production of recombinant plasmid DNA. The highlighted issues will be beneficial in characterizing and manufacturing plasmid DNA for save clinical use. Manipulation of regulatory units of plasmid will have impact towards addressing the safety concerns raised in human vaccine applications. The gene revolution with plasmid DNA by alteration of their plasmid and production host genetics will be promising for safe delivery and obtaining efficient outcomes.

  7. Development of a recombinant DNA assay system for the detection of genetic change in astronauts' cells

    SciTech Connect

    Atchley, S.V.; Chen, D.J.C.; Strniste, G.F.; Walters, R.A.; Moyzis, R.K.

    1984-01-01

    We are developing a new recombinant DNA system for the detection and measurement of genetic change in humans caused by exposure to low level ionizing radiation. A unique feature of the method is the use of cloned repetitive DNA probes to assay human DNA for structural changes during or after irradiation. Repetitive sequences exist in different families. Collectively they constitute over 25% of the DNA in a human cell. Repeat families have between 10 and 500,000 members. We have constructed repetitive DNA sequence libraries using recombinant DNA techniques. From these libraries we have isolated and characterized individual repeats comprising 75 to 90% of the mass of human repetitive DNA. Repeats used in our assay system exist in tandem arrays in the genome. Perturbation of these sequences in a cell, followed by detection with a repeat probe, produces a new, multimeric ''ladder'' pattern on an autoradiogram. The repeat probe used in our initial study is complementary to 1% of human DNA. Therefore, the sensitivity of this method is several orders of magnitude better than existing assays. Preliminary evidence from human skin cells exposed to acute, low-dose x-ray treatments indicates that DNA is affected at a dose as low as 5R. The radiation doses used in this system are well within the range of doses received by astronauts during spaceflight missions. Due to its small material requirements, this technique could easily be adapted for use in space. 16 refs., 1 fig.

  8. Remodeling and Control of Homologous Recombination by DNA Helicases and Translocases that Target Recombinases and Synapsis

    PubMed Central

    Northall, Sarah J.; Ivančić-Baće, Ivana; Soultanas, Panos; Bolt, Edward L.

    2016-01-01

    Recombinase enzymes catalyse invasion of single-stranded DNA (ssDNA) into homologous duplex DNA forming “Displacement loops” (D-loops), a process called synapsis. This triggers homologous recombination (HR), which can follow several possible paths to underpin DNA repair and restart of blocked and collapsed DNA replication forks. Therefore, synapsis can be a checkpoint for controlling whether or not, how far, and by which pathway, HR proceeds to overcome an obstacle or break in a replication fork. Synapsis can be antagonized by limiting access of a recombinase to ssDNA and by dissociation of D-loops or heteroduplex formed by synapsis. Antagonists include DNA helicases and translocases that are identifiable in eukaryotes, bacteria and archaea, and which target synaptic and pre-synaptic DNA structures thereby controlling HR at early stages. Here we survey these events with emphasis on enabling DNA replication to be resumed from sites of blockage or collapse. We also note how knowledge of anti-recombination activities could be useful to improve efficiency of CRISPR-based genome editing. PMID:27548227

  9. Distribution of Exchanges upon Homologous Recombination of Exogenous DNA in Xenopus Laevis Oocytes

    PubMed Central

    Carroll, D.; Lehman, C. W.; Jeong-Yu, S.; Dohrmann, P.; Dawson, R. J.; Trautman, J. K.

    1994-01-01

    Homologous recombination between DNA molecules injected into Xenopus oocyte nuclei was investigated by examining the recovery of information from differentially marked parental sequences. The injected recombination substrate was a linear DNA with terminal direct repeats of 1246 bp; one repeat differed from the other by eight single base-pair substitutions, distributed throughout the region of homology, each of which created or destroyed a restriction enzyme site. Recombination products were recovered and analyzed for their content of the diagnostic sites, either directly by Southern blot-hybridization or after cloning in bacteria. The majority (76%) of the cloned products appeared to be the result of simple exchanges-i.e., there was one sharp transition from sequences derived from one parent to sequences derived from the other. These simple exchanges were concentrated near the ends of the homologous interval and, thus, near the sites of the original molecular ends. Placing marked sites on only one side of the homologous overlap showed that marker recovery was governed largely by the positions of the molecular ends and not by the markers themselves. When a terminal nonhomology was present at one end of the substrate, the yield of recombinants was sharply decreased, but the pattern of exchanges was not affected, suggesting that products from end-blocked substrates arise by the same recombination pathway. Because of considerable evidence supporting a nonconservative, resection-annealing mechanism for recombination in oocytes, we interpret the distribution of exchanges as resulting from long-patch repair of extensive heteroduplex intermediates. PMID:7828826

  10. BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure.

    PubMed

    Yang, Haijuan; Jeffrey, Philip D; Miller, Julie; Kinnucan, Elspeth; Sun, Yutong; Thoma, Nicolas H; Zheng, Ning; Chen, Phang-Lang; Lee, Wen-Hwa; Pavletich, Nikola P

    2002-09-13

    Mutations in the BRCA2 (breast cancer susceptibility gene 2) tumor suppressor lead to chromosomal instability due to defects in the repair of double-strand DNA breaks (DSBs) by homologous recombination, but BRCA2's role in this process has been unclear. Here, we present the 3.1 angstrom crystal structure of a approximately 90-kilodalton BRCA2 domain bound to DSS1, which reveals three oligonucleotide-binding (OB) folds and a helix-turn-helix (HTH) motif. We also (i) demonstrate that this BRCA2 domain binds single-stranded DNA, (ii) present its 3.5 angstrom structure bound to oligo(dT)9, (iii) provide data that implicate the HTH motif in dsDNA binding, and (iv) show that BRCA2 stimulates RAD51-mediated recombination in vitro. These findings establish that BRCA2 functions directly in homologous recombination and provide a structural and biochemical basis for understanding the loss of recombination-mediated DSB repair in BRCA2-associated cancers.

  11. Roles of RECQ helicases in recombination based DNA repair, genomic stability and aging

    PubMed Central

    Singh, Dharmendra Kumar; Ahn, Byungchan

    2009-01-01

    The maintenance of the stability of genetic material is an essential feature of every living organism. Organisms across all kingdoms have evolved diverse and highly efficient repair mechanisms to protect the genome from deleterious consequences of various genotoxic factors that might tend to destabilize the integrity of the genome in each generation. One such group of proteins that is actively involved in genome surveillance is the RecQ helicase family. These proteins are highly conserved DNA helicases, which have diverse roles in multiple DNA metabolic processes such as DNA replication, recombination and DNA repair. In humans, five RecQ helicases have been identified and three of them namely, WRN, BLM and RecQL4 have been linked to genetic diseases characterized by genome instability, premature aging and cancer predisposition. This helicase family plays important roles in various DNA repair pathways including protecting the genome from illegitimate recombination during chromosome segregation in mitosis and assuring genome stability. This review mainly focuses on various roles of human RecQ helicases in the process of recombination-based DNA repair to maintain genome stability and physiological consequences of their defects in the development of cancer and premature aging. PMID:19083132

  12. Cre-dependent DNA recombination activates a STING-dependent innate immune response

    PubMed Central

    Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W. Samantha N.; Cain, Jason E.; Behlke, Mark A.; Gough, Daniel J.; G. Williams, Bryan R.; Hornung, Veit; Gantier, Michael P.

    2016-01-01

    Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell–cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376

  13. Roles of RECQ helicases in recombination based DNA repair, genomic stability and aging.

    PubMed

    Singh, Dharmendra Kumar; Ahn, Byungchan; Bohr, Vilhelm A

    2009-06-01

    The maintenance of the stability of genetic material is an essential feature of every living organism. Organisms across all kingdoms have evolved diverse and highly efficient repair mechanisms to protect the genome from deleterious consequences of various genotoxic factors that might tend to destabilize the integrity of the genome in each generation. One such group of proteins that is actively involved in genome surveillance is the RecQ helicase family. These proteins are highly conserved DNA helicases, which have diverse roles in multiple DNA metabolic processes such as DNA replication, recombination and DNA repair. In humans, five RecQ helicases have been identified and three of them namely, WRN, BLM and RecQL4 have been linked to genetic diseases characterized by genome instability, premature aging and cancer predisposition. This helicase family plays important roles in various DNA repair pathways including protecting the genome from illegitimate recombination during chromosome segregation in mitosis and assuring genome stability. This review mainly focuses on various roles of human RecQ helicases in the process of recombination-based DNA repair to maintain genome stability and physiological consequences of their defects in the development of cancer and premature aging.

  14. Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae

    PubMed Central

    Pâques, Frédéric; Haber, James E.

    1999-01-01

    The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination. PMID:10357855

  15. Recombinant covalently closed circular hepatitis B virus DNA induces prolonged viral persistence in immunocompetent mice.

    PubMed

    Qi, Zhihua; Li, Gaiyun; Hu, Hao; Yang, Chunhui; Zhang, Xiaoming; Leng, Qibin; Xie, Youhua; Yu, Demin; Zhang, Xinxin; Gao, Yueqiu; Lan, Ke; Deng, Qiang

    2014-07-01

    It remains crucial to develop a laboratory model for studying hepatitis B virus (HBV) chronic infection. We hereby produced a recombinant covalently closed circular DNA (rcccDNA) in view of the key role of cccDNA in HBV persistence. A loxP-chimeric intron was engineered into a monomeric HBV genome in a precursor plasmid (prcccDNA), which was excised using Cre/loxP-mediated DNA recombination into a 3.3-kb rcccDNA in the nuclei of hepatocytes. The chimeric intron was spliced from RNA transcripts without interrupting the HBV life cycle. In cultured hepatoma cells, cotransfection of prcccDNA and pCMV-Cre (encoding Cre recombinase) resulted in accumulation of nuclear rcccDNA that was heat stable and epigenetically organized as a minichromosome. A mouse model of HBV infection was developed by hydrodynamic injection of prcccDNA. In the presence of Cre recombinase, rcccDNA was induced in the mouse liver with effective viral replication and expression, triggering a compromised T-cell response against HBV. Significant T-cell hyporesponsiveness occurred in mice receiving 4 μg prcccDNA, resulting in prolonged HBV antigenemia for up to 9 weeks. Persistent liver injury was observed as elevated alanine transaminase activity in serum and sustained inflammatory infiltration in the liver. Although a T-cell dysfunction was induced similarly, mice injected with a plasmid containing a linear HBV replicon showed rapid viral clearance within 2 weeks. Collectively, our study provides an innovative approach for producing a cccDNA surrogate that established HBV persistence in immunocompetent mice. It also represents a useful model system in vitro and in vivo for evaluating antiviral treatments against HBV cccDNA. Importance: (i) Unlike plasmids that contain a linear HBV replicon, rcccDNA established HBV persistence with sustained liver injury in immunocompetent mice. This method could be a prototype for developing a mouse model of chronic HBV infection. (ii) An exogenous intron was

  16. Diversity and recombination of dispersed ribosomal DNA and protein coding genes in microsporidia.

    PubMed

    Ironside, Joseph Edward

    2013-01-01

    Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and should therefore exhibit reduced genetic diversity due to a lack of recombination. Here, DNA sequences are compared between microsporidia with different life cycles in order to determine the effects of concerted evolution and sexual reproduction upon the diversity of rDNA and protein coding genes. Comparisons of cloned rDNA sequences between microsporidia of the genus Nosema with different life cycles provide evidence of intragenomic variability coupled with strong purifying selection. This suggests a birth and death process of evolution. However, some concerted evolution is suggested by clustering of rDNA sequences within species. Variability of protein-coding sequences indicates that considerable intergenomic variation also occurs between microsporidian cells within a single host. Patterns of variation in microsporidian DNA sequences indicate that additional diversity is generated by intragenomic and/or intergenomic recombination between sequence variants. The discovery of intragenomic variability coupled with strong purifying selection in microsporidian rRNA sequences supports the hypothesis that concerted evolution is reduced when copies of a gene are dispersed rather than repeated tandemly. The presence of intragenomic variability also renders the use of rDNA sequences for barcoding microsporidia questionable. Evidence of recombination in the single-copy genes of putatively asexual microsporidia suggests that these species may undergo cryptic sexual reproduction, a

  17. Diversity and Recombination of Dispersed Ribosomal DNA and Protein Coding Genes in Microsporidia

    PubMed Central

    Ironside, Joseph Edward

    2013-01-01

    Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and should therefore exhibit reduced genetic diversity due to a lack of recombination. Here, DNA sequences are compared between microsporidia with different life cycles in order to determine the effects of concerted evolution and sexual reproduction upon the diversity of rDNA and protein coding genes. Comparisons of cloned rDNA sequences between microsporidia of the genus Nosema with different life cycles provide evidence of intragenomic variability coupled with strong purifying selection. This suggests a birth and death process of evolution. However, some concerted evolution is suggested by clustering of rDNA sequences within species. Variability of protein-coding sequences indicates that considerable intergenomic variation also occurs between microsporidian cells within a single host. Patterns of variation in microsporidian DNA sequences indicate that additional diversity is generated by intragenomic and/or intergenomic recombination between sequence variants. The discovery of intragenomic variability coupled with strong purifying selection in microsporidian rRNA sequences supports the hypothesis that concerted evolution is reduced when copies of a gene are dispersed rather than repeated tandemly. The presence of intragenomic variability also renders the use of rDNA sequences for barcoding microsporidia questionable. Evidence of recombination in the single-copy genes of putatively asexual microsporidia suggests that these species may undergo cryptic sexual reproduction, a

  18. A Citizen Court in the Recombinant DNA debate.

    ERIC Educational Resources Information Center

    Krinsky, Sheldon

    1978-01-01

    Harvard scientists were planning DNA experiments which required special facilities. A citizen panel was formed to look into the adequacy of federal safety guidelines for the community. Describes the review process and discusses the concept of a citizen court to resolve such technical controversies. (GA)

  19. Depletion of Key Meiotic Genes and Transcriptome-Wide Abiotic Stress Reprogramming Mark Early Preparatory Events Ahead of Apomeiotic Transition

    PubMed Central

    Shah, Jubin N.; Kirioukhova, Olga; Pawar, Pallavi; Tayyab, Muhammad; Mateo, Juan L.; Johnston, Amal J.

    2016-01-01

    Molecular dissection of apomixis – an asexual reproductive mode – is anticipated to solve the enigma of loss of meiotic sex, and to help fixing elite agronomic traits. The Brassicaceae genus Boechera comprises of both sexual and apomictic species, permitting comparative analyses of meiotic circumvention (apomeiosis) and parthenogenesis. Whereas previous studies reported local transcriptome changes during these events, it remained unclear whether global changes associated with hybridization, polyploidy and environmental adaptation that arose during evolution of Boechera might serve as (epi)genetic regulators of early development prior apomictic initiation. To identify these signatures during vegetative stages, we compared seedling RNA-seq transcriptomes of an obligate triploid apomict and a diploid sexual, both isolated from a drought-prone habitat. Uncovered were several genes differentially expressed between sexual and apomictic seedlings, including homologs of meiotic genes ASYNAPTIC 1 (ASY1) and MULTIPOLAR SPINDLE 1 (MPS1) that were down-regulated in apomicts. An intriguing class of apomict-specific deregulated genes included several NAC transcription factors, homologs of which are known to be transcriptionally reprogrammed during abiotic stress in other plants. Deregulation of both meiotic and stress-response genes during seedling stages might possibly be important in preparation for meiotic circumvention, as similar transcriptional alteration was discernible in apomeiotic floral buds too. Furthermore, we noted that the apomict showed better tolerance to osmotic stress in vitro than the sexual, in conjunction with significant upregulation of a subset of NAC genes. In support of the current model that DNA methylation epigenetically regulates stress, ploidy, hybridization and apomixis, we noted that ASY1, MPS1 and NAC019 homologs were deregulated in Boechera seedlings upon DNA demethylation, and ASY1 in particular seems to be repressed by global DNA

  20. Artificial restriction DNA cutters to promote homologous recombination in human cells.

    PubMed

    Katada, Hitoshi; Komiyama, Makoto

    2011-02-01

    Homologous recombination is almost the only way to modify the genome in a predetermined fashion, despite its quite low frequency in mammalian cells. It has been already reported that the frequency of this biological process can be notably increased by inducing a double strand break (DSB) at target site. This article presents completely chemistry-based artificial restriction DNA cutter (ARCUT) for the promotion of homologous recombination in human cells. This cutter is composed of Ce(IV)/EDTA complex (molecular scissors) and two strands of peptide nucleic acid (PNA), and contains no proteins. Its scission site in the genome is determined simply by Watson-Crick rule so that ARCUT for desired homologous recombination is easily and straightforwardly designed and synthesized. The site-specificity of the scission is high enough to cut human genome at one target site. The DSB induced by this cutter is satisfactorily recognized by the repair system in human cells and promotes the targeted homologous recombination.

  1. The "Frankenplasmid" Lab: An Investigative Exercise for Teaching Recombinant DNA Methods

    ERIC Educational Resources Information Center

    Dean, Derek M.; Wilder, Jason A.

    2011-01-01

    We describe an investigative laboratory module designed to give college undergraduates strong practical and theoretical experience with recombinant DNA methods within 3 weeks. After deducing restriction enzyme maps for two different plasmids, students ligate the plasmids together in the same reaction, transform "E. coli" with this mixture of…

  2. Personal reflections on the origins and emergence of recombinant DNA technology.

    PubMed

    Berg, Paul; Mertz, Janet E

    2010-01-01

    The emergence of recombinant DNA technology occurred via the appropriation of known tools and procedures in novel ways that had broad applications for analyzing and modifying gene structure and organization of complex genomes. Although revolutionary in their impact, the tools and procedures per se were not revolutionary. Rather, the novel ways in which they were applied was what transformed biology.

  3. Government Regulation of the Pursuit of Knowledge: The Recombinant DNA Controversy.

    ERIC Educational Resources Information Center

    Berger, Richard G.

    1978-01-01

    Government regulation of recombinant DNA research is addressed. Issues discussed include the potential of such research; National Institutes of Health guidelines; federal, state, and local regulation; the controversy over self-regulation; first amendment protection for scientific research; and problems in drafting legislation. (JMD)

  4. Are High School Students Ready for Recombinant DNA?: The UOP Experience.

    ERIC Educational Resources Information Center

    Minch, Michael J.

    1989-01-01

    Discusses a three-week summer college honors course for talented high school juniors with three exams, lab six days a week, a research paper, field trips, and student panel discussions. Presents an overview of the course. Describes the lab which uses "E. coli" for DNA recombination. (MVL)

  5. [SPO11: an activity that promotes DNA breaks required for meiosis].

    PubMed

    Baudat, Frédéric; de Massy, Bernard

    2004-02-01

    Recombination between homologous chromosomes during meiosis is an essential process, which mechanistical function is to ensure the reductional segregation of chromosomes at the first meiotic division. SPO11, one of the key genes directly involved in this process, has been at the origin of considerable interest for the past five years, for several reasons. First, Spo11 is responsible for the initiation of meiotic recombination through the formation of DNA double-strand breaks by a type II DNA topoisomerase-like activity. Moreover, Spo11, and its function, have been conserved through evolution, from yeasts to human, as demonstrated by the identification of members of the Spo11 protein family and the analyses of corresponding mutants. Indeed, for every eukaryote that has been tested, spo11 mutants are deficient for meiotic recombination and are partially or completely sterile. Depending on the species, this reduced fertility reflects either a defect in chromosome segregation, or an arrest response in germ cell differentiation. Similarities and differences from species to species uncover a complex set of regulations that coordinate recombination with other events of meiotic prophase, such as chromosome pairing and meiotic cell cycle.

  6. Preferential repair of UV damage in highly transcribed DNA diminishes UV-induced intrachromosomal recombination in mammalian cells.

    PubMed Central

    Deng, W P; Nickoloff, J A

    1994-01-01

    The relationships among transcription, recombination, DNA damage, and repair in mammalian cells were investigated. We monitored the effects of transcription on UV-induced intrachromosomal recombination between neomycin repeats including a promoterless allele and an inducible heteroallele regulated by the mouse mammary tumor virus promoter. Although transcription and UV light separately stimulated recombination, increasing transcription levels reduced UV-induced recombination. Preferential repair of UV damage in transcribed strands was shown in highly transcribed DNA, suggesting that recombination is stimulated by unrepaired UV damage and that increased DNA repair in highly transcribed alleles removes recombinogenic lesions. This study indicates that the genetic consequences of DNA damage depend on transcriptional states and provides a basis for understanding tissue- and gene-specific responses to DNA-damaging agents. Images PMID:8264606

  7. The role of DNA repair genes in recombination between repeated sequences in yeast.

    PubMed

    Liefshitz, B; Parket, A; Maya, R; Kupiec, M

    1995-08-01

    The presence of repeated sequences in the genome represents a potential source of karyotypic instability. Genetic control of recombination is thus important to preserve the integrity of the genome. To investigate the genetic control of recombination between repeated sequences, we have created a series of isogenic strains in which we could assess the role of genes involved in DNA repair in two types of recombination: direct repeat recombination and ectopic gene conversion. Naturally occurring (Ty elements) and artificially constructed repeats could be compared in the same cell population. We have found that direct repeat recombination and gene conversion have different genetic requirements. The role of the RAD51, RAD52, RAD54, RAD55, and RAD57 genes, which are involved in recombinational repair, was investigated. Based on the phenotypes of single and double mutants, these genes can be divided into three functional subgroups: one composed of RAD52, a second one composed of RAD51 and RAD54, and a third one that includes the RAD55 and RAD57 genes. Among seven genes involved in excision repair tested, only RAD1 and RAD10 played a role in the types of recombination studied. We did not detect a differential effect of any rad mutation on Ty elements as compared to artificially constructed repeats.

  8. The Arabidopsis DNA mismatch repair gene PMS1 restricts somatic recombination between homeologous sequences.

    PubMed

    Li, Liangliang; Dion, Eric; Richard, Gabriel; Domingue, Olivier; Jean, Martine; Belzile, François J

    2009-04-01

    The eukaryotic DNA mismatch repair (MMR) system contributes to maintaining the fidelity of genetic information by correcting replication errors and preventing illegitimate recombination events. This study aimed to examine the function(s) of the Arabidopsis thaliana PMS1 gene (AtPMS1), one of three homologs of the bacterial MutL gene in plants. Two independent mutant alleles (Atpms1-1 and Atpms1-2) were obtained and one of these (Atpms1-1) was studied in detail. The mutant exhibited a reduction in seed set and a bias against the transmission of the mutant allele. Somatic recombination, both homologous and homeologous, was examined using a set of reporter constructs. Homologous recombination remained unchanged in the mutant while homeologous recombination was between 1.7- and 4.8-fold higher than in the wild type. This increase in homeologous recombination frequency was not correlated with the degree of sequence divergence. In RNAi lines, a range of increases in homeologous recombination were observed with two lines showing a 3.3-fold and a 3.6-fold increase. These results indicate that the AtPMS1 gene contributes to an antirecombination activity aimed at restricting recombination between diverged sequences.

  9. The effects of translocations on recombination frequency in Caenorhabditis elegans.

    PubMed

    McKim, K S; Howell, A M; Rose, A M

    1988-12-01

    In the nematode Caenorhabditis elegans, recombination suppression in translocation heterozygotes is severe and extensive. We have examined the meiotic properties of two translocations involving chromosome I, szT1(I;X) and hT1(I;V). No recombination was observed in either of these translocation heterozygotes along the left (let-362-unc-13) 17 map units of chromosome I. Using half-translocations as free duplications, we mapped the breakpoints of szT1 and hT1. The boundaries of crossover suppression coincided with the physical breakpoints. We propose that DNA sequences at the right end of chromosome I facilitate pairing and recombination. We use the data from translocations of other chromosomes to map the location of pairing sites on four other chromosomes. hT1 and szT1 differed markedly in their effect on recombination adjacent to the crossover suppressed region. hT1 had no effect on recombination in the adjacent interval. In contrast, the 0.8 map unit interval immediately adjacent to the szT1(I;X) breakpoint on chromosome I increased to 2.5 map units in translocation heterozygotes. This increase occurs in a chromosomal interval which can be expanded by treatment with radiation. These results are consistent with the suggestion that the szT1(I) breakpoint is in a region of DNA in which meiotic recombination is suppressed relative to the genomic average. We propose that DNA sequences disrupted by the szT1 translocation are responsible for determining the frequency of meiotic recombination in the vicinity of the breakpoint.

  10. Absence of extensive recombination between inter- and intraspecies mitochondrial DNA in mammalian cells.

    PubMed

    Hayashi, J; Tagashira, Y; Yoshida, M C

    1985-10-01

    Recombination of mammalian mitochondrial DNA (mtDNA) was examined using mouse X rat somatic cell hybrid clones and rat cybrid clones. The mouse X rat hybrids were isolated by fusion of chloramphenicol-sensitive (CAPs) mouse and CAP-resistant (CAPr) rat cells. The rat cybrids were isolated by fusion of rat cells with type B mtDNA and enucleated cells with type A mtDNA. Genetic and physical analyses showed that the mtDNAs of the hybrids and cybrids were simple mixtures of the two parental mtDNAs except in the following two cases: One was subclone H2-9 of mouse X rat hybrids, which was CAPr even though mtDNA from the CAPs mouse parent was predominantly retained. The other was rat cybrid subclones, Y12-24 and -61, which showed specific loss of one Hinf I fragment of type B mtDNA, B10. These observations suggest that, in contrast to the case with plant mtDNA, recombination of mammalian mtDNA occurs rarely, if at all.

  11. Quantitation of the residual DNA from rice-derived recombinant human serum albumin.

    PubMed

    Chen, Zhen; Dai, Huixia; Liu, Zhenwei; Zhang, Liping; Pang, Jianlei; Ou, Jiquan; Yang, Daichang

    2014-04-01

    Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2×10(4) to 0.2pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.

  12. Neutralizing antibodies respond to a bivalent dengue DNA vaccine or/and a recombinant bivalent antigen.

    PubMed

    Zhang, Zhi-Shan; Weng, Yu-Wei; Huang, Hai-Long; Zhang, Jian-Ming; Yan, Yan-Sheng

    2015-02-01

    There is currently no effective vaccine to prevent dengue infection, despite the existence of multiple studies on potential methods of immunization. The aim of the present study was to explore the effect of DNA and/or recombinant protein on levels of neutralizing antibodies. For this purpose, envelope domain IIIs of dengue serotypes 1 and 2 (DEN-1/2)were spliced by a linker (Gly‑Gly‑Ser‑Gly‑Ser)3 and cloned into the prokaryotic expression plasmid pET30a (+) and eukaryotic vector pcDNA3.1 (+). The chimeric bivalent protein was expressed in Escherichia coli, and one‑step purification by high‑performance liquid chromatography was conducted. Protein expression levels of the DNA plasmid were tested in BHK‑21 cells by indirect immunofluorescent assay. In order to explore a more effective immunization strategy and to develop neutralizing antibodies against the two serotypes, mice were inoculated with recombinant bivalent protein, the DNA vaccine, or the two given simultaneously. Presence of the specific antibodies was tested by ELISA and the presence of the neutralizing antibodies was determined by plaque reduction neutralization test. Results of the analysis indicated that the use of a combination of DNA and protein induced significantly higher titers of neutralizing antibodies against either DEN‑1 or DEN‑2 (1:64.0 and 1:76.1, respectively) compared with the DNA (1:24.7 and 1:26.9, DEN‑1 and DEN‑2, respectively) or the recombinant protein (1:34.9 and 1:45.3 in DEN‑1 and DEN‑2, respectively). The present study demonstrated that the combination of recombinant protein and DNA as an immunization strategy may be an effective method for the development of a vaccine to prevent dengue virus infection.

  13. DNA End Resection: Nucleases Team Up with the Right Partners to Initiate Homologous Recombination.

    PubMed

    Cejka, Petr

    2015-09-18

    The repair of DNA double-strand breaks by homologous recombination commences by nucleolytic degradation of the 5'-terminated strand of the DNA break. This leads to the formation of 3'-tailed DNA, which serves as a substrate for the strand exchange protein Rad51. The nucleoprotein filament then invades homologous DNA to drive template-directed repair. In this review, I discuss mainly the mechanisms of DNA end resection in Saccharomyces cerevisiae, which includes short-range resection by Mre11-Rad50-Xrs2 and Sae2, as well as processive long-range resection by Sgs1-Dna2 or Exo1 pathways. Resection mechanisms are highly conserved between yeast and humans, and analogous machineries are found in prokaryotes as well.

  14. Rec8 guides canonical Spo11 distribution along yeast meiotic chromosomes.

    PubMed

    Kugou, Kazuto; Fukuda, Tomoyuki; Yamada, Shintaro; Ito, Masaru; Sasanuma, Hiroyuki; Mori, Saori; Katou, Yuki; Itoh, Takehiko; Matsumoto, Kouji; Shibata, Takehiko; Shirahige, Katsuhiko; Ohta, Kunihiro

    2009-07-01

    Spo11-mediated DNA double-strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To elucidate this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thus, we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation.

  15. Homologous recombination intermediates between two duplex DNA catalysed by human cell extracts.

    PubMed Central

    Lopez, B; Rousset, S; Coppey, J

    1987-01-01

    Using as substrates, 1: the replicative form (RF) of phage M13 mp8 in which the reading frame of the lac Z' gene was disrupted by insertion of an octonucleotide, and 2: a restriction fragment one kb long, containing the functional lac Z' gene (isolated from wild type M13 mp8), we show that nuclear extracts from human cells (3 lines tested) promote the targeted replacement of the altered sequence by the functional one. Following incubation with the extracts, the DNA's were introduced in JM 109 bacteria (rec A- and lac Z'-) which were grown in presence of a colorimetric indicator of beta-galactosidase activity. Homologous recombination gives rise to the genotypical modification: lac Z'+ instead of lac Z'- in the bacteriophage DNA. This is revealed by phenotypical expression of the lac Z' gene product in replicating bacteriophage, i.e. the formation of blue instead of white plaques. The frequency of recombination (blue/total plaques) is increased by a factor of 50-80 as a function of protein concentration and of incubation time. The maximal frequency observed is 5 X 10(-5). There is no increase over the background when extracts are boiled. Electrophoresis and electron microscopy of DNA's incubated with the extracts show the formation of recombination intermediates with single strand exchange. Restriction analysis of recombined DNA confirms that the process corresponds to targeted sequence exchange. These data allow to propose three steps for homologous recombination between two duplex DNA's: i) unpairing of the two duplexes; ii) single-strand exchange and synaptic pairing; iii) resolution of the cross-junctions. The three steps correspond to those predicted by the gene conversion model of Holliday. Images PMID:3302944

  16. The cell pole: The site of cross talk between the DNA uptake and genetic recombination machinery

    PubMed Central

    Kidane, Dawit; Ayora, Silvia; Sweasy, Joann; Graumann, Peter L.; Alonso, Juan C.

    2012-01-01

    Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as “guardians”, protect ssDNA from degradation and limit the RecA recombinase loading. Then, the “mediators” overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by “modulators”, catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or “resolver” cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the “rescuers” will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective. PMID:23046409

  17. The homologous recombination system of Ustilago maydis.

    PubMed

    Holloman, William K; Schirawski, Jan; Holliday, Robin

    2008-08-01

    Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of U. maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins.

  18. The homologous recombination system of Ustilago maydis

    PubMed Central

    Holloman, William K.; Schirawski, Jan; Holliday, Robin

    2008-01-01

    Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of Ustilago maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins. PMID:18502156

  19. Molecular analysis of recombination in a family with Duchenne muscular dystrophy and a large pericentric X chromosome inversion

    SciTech Connect

    Shashi, V.; Golden, W.L.; Allinson, P.S.

    1996-06-01

    It has been demonstrated in animal studies that, in animals heterozygous for pericentric chromosomal inversions, loop formation is greatly reduced during meiosis. This results in absence of recombination within the inverted segment, with recombination seen only outside the inversion. A recent study in yeast has shown that telomeres, rather than centromeres, lead in chromosome movement just prior to meiosis and may be involved in promoting recombination. We studied by cytogenetic analysis and DNA polymorphisms the nature of meiotic recombination in a three-generation family with a large pericentric X chromosome inversion, inv(X)(p21.1q26), in which Duchenne muscular dystrophy (DMD) was cosegregating with the inversion. On DNA analysis there was no evidence of meiotic recombination between the inverted and normal X chromosomes in the inverted segment. Recombination was seen at the telomeric regions, Xp22 and Xq27-28. No deletion or point mutation was found on analysis of the DMD gene. On the basis of the FISH results, we believe that the X inversion is the mutation responsible for DMD in this family. Our results indicate that (1) pericentric X chromosome inversions result in reduction of recombination between the normal and inverted X chromosomes; (2) meiotic X chromosome pairing in these individuals is likely initiated at the telomeres; and (3) in this family DMD is caused by the pericentric inversion. 50 refs., 7 figs., 1 tab.

  20. Molecular analysis of recombination in a family with Duchenne muscular dystrophy and a large pericentric X chromosome inversion.

    PubMed Central

    Shashi, V.; Golden, W. L.; Allinson, P. S.; Blanton, S. H.; von Kap-Herr, C.; Kelly, T. E.

    1996-01-01

    It has been demonstrated in animal studies that, in animals heterozygous for pericentric chromosomal inversions, loop formation is greatly reduced during meiosis. This results in absence of recombination within the inverted segment, with recombination seen only outside the inversion. A recent study in yeast has shown that telomeres, rather than centromeres, lead in chromosome movement just prior to meiosis and may be involved in promoting recombination. We studied by cytogenetic analysis and DNA polymorphisms the nature of meiotic recombination in a three-generation family with a large pericentric X chromosome inversion, inv(X)(p21.1q26), in which Duchenne muscular dystrophy (DMD) was cosegregating with the inversion. On DNA analysis there was no evidence of meiotic recombination between the inverted and normal X chromosomes in the inverted segment. Recombination was seen at the telomeric regions, Xp22 and Xq27-28. No deletion or point mutation was found on analysis of the DMD gene. On the basis of the FISH results, we believe that the X inversion is the mutation responsible for DMD in this family. Our results indicate that (1) pericentric X chromosome inversions result in reduction of recombination between the normal and inverted X chromosomes; (2) meiotic X chromosome pairing in these individuals is likely initiated at the telomeres; and (3) in this family DMD is caused by the pericentric inversion. Images Figure 2 Figure 5 Figure 6 Figure 7 PMID:8651300

  1. Recombinational DNA repair in a cellular context: a search for the homology search.

    PubMed

    Weiner, Allon; Zauberman, Nathan; Minsky, Abraham

    2009-10-01

    Double-strand DNA breaks (DSBs) are the most detrimental lesion that can be sustained by the genetic complement, and their inaccurate mending can be just as damaging. According to the consensual view, precise DSB repair relies on homologous recombination. Here, we review studies on DNA repair, chromatin diffusion and chromosome confinement, which collectively imply that a genome-wide search for a homologous template, generally thought to be a pivotal stage in all homologous DSB repair pathways, is improbable. The implications of this assertion for the scope and constraints of DSB repair pathways and for the ability of diverse organisms to cope with DNA damage are discussed.

  2. Oocyte shuttle, a recombinant protein transporting donor DNA into the Xenopus oocyte in situ.

    PubMed

    Rungger, Duri; Muster, Lisbeth; Georgiev, Oleg; Rungger-Brändle, Elisabeth

    2017-02-15

    The newly developed oocyte shuttle protein contains a streptavidin moiety that tightly binds biotinylated DNA. Injected intravenously into adult Xenopus females, the protein-DNA complex is rapidly transported through the bloodstream and, within the ovary, the vitellogenin ligand present in the protein binds to the receptors at the surface of the oocytes. The bound complex is internalized and translocates into the oocyte nucleus thanks to an SV40 nuclear localization signal, enhanced by an adjacent casein kinase phosphorylation site. Functioning of the shuttle protein is documented by transporting DNA molecules that, upon intramolecular homologous recombination within the oocyte nucleus, express easily traceable markers such as green fluorescence or tetracycline resistance.

  3. Conditional mutagenesis of the genome using site-specific DNA recombination.

    PubMed

    Ohtsubo, Kazuaki; Marth, Jamey D

    2007-07-01

    INTRODUCTIONAltering the genome of intact cells and organisms by site-specific DNA recombination has become an important gene-transfer methodology. DNA modifications produced by gene transfer and homologous recombination are typically static once integrated among target cell chromosomes. In contrast, the inclusion of exogenous recombinase target sequences within transferred DNA segments allows subsequent modifications to previously altered genomic structure that increase the utility of gene transfer and enhance experimental design. Creating tissue- and cell-type-specific genetic lesions in animal models, indelibly marking progenitors for cell fate mapping, inducing large-scale chromosomal rearrangements, and complementing gene defects in studies of phenotypic maintenance and reversion are all possible by directing recombinase expression using gene transfer among experimentally modified genomes. Moreover, this approach is effective in providing controlled data establishing genotype-phenotype relationships and allows for the excision of introduced marker genes that can affect neighboring chromatin structure and function. Although early work involved the yeast Flp recombinase, most studies in mammalian systems have used the Cre recombinase derived from bacteriophage P1. Both enzymes are members of the integrase family of recombinases but bind to distinct DNA target signals. Cre recombinase operates on the 34-bp loxP sequence and, like Flp, performs conservative recombination involving DNA segments positioned among these target sites.

  4. Purification and Characterization of a DNA-Binding Recombinant PREP1:PBX1 Complex

    PubMed Central

    Mathiasen, Lisa; Bruckmann, Chiara; Pasqualato, Sebastiano; Blasi, Francesco

    2015-01-01

    Human PREP1 and PBX1 are homeodomain transcriptional factors, whose biochemical and structural characterization has not yet been fully described. Expression of full-length recombinant PREP1 (47.6 kDa) and PBX1 (46.6 kDa) in E. coli is difficult because of poor yield, high instability and insufficient purity, in particular for structural studies. We cloned the cDNA of both proteins into a dicistronic vector containing an N-terminal glutathione S-transferase (GST) tag and co-expressed and co-purified a stable PBX1:PREP1 complex. For structural studies, we produced two C-terminally truncated complexes that retain their ability to bind DNA and are more stable than the full-length proteins through various purification steps. Here we report the production of large amounts of soluble and pure recombinant human PBX1:PREP1 complex in an active form capable of binding DNA. PMID:25856340

  5. Purification and characterization of a DNA-binding recombinant PREP1:PBX1 complex.

    PubMed

    Mathiasen, Lisa; Bruckmann, Chiara; Pasqualato, Sebastiano; Blasi, Francesco

    2015-01-01

    Human PREP1 and PBX1 are homeodomain transcriptional factors, whose biochemical and structural characterization has not yet been fully described. Expression of full-length recombinant PREP1 (47.6 kDa) and PBX1 (46.6 kDa) in E. coli is difficult because of poor yield, high instability and insufficient purity, in particular for structural studies. We cloned the cDNA of both proteins into a dicistronic vector containing an N-terminal glutathione S-transferase (GST) tag and co-expressed and co-purified a stable PBX1:PREP1 complex. For structural studies, we produced two C-terminally truncated complexes that retain their ability to bind DNA and are more stable than the full-length proteins through various purification steps. Here we report the production of large amounts of soluble and pure recombinant human PBX1:PREP1 complex in an active form capable of binding DNA.

  6. Plasmid-Chromosome Recombination of Irradiated Shuttle Vector DNA in African Green Monkey Kidney Cells.

    NASA Astrophysics Data System (ADS)

    Mudgett, John Stuart

    1987-09-01

    An autonomously replicating shuttle vector was used to investigate the enhancement of plasmid-chromosome recombination in mammalian host cells by ultraviolet light and gamma radiation. Sequences homologous to the shuttle vector were stably inserted into the genome of African Green Monkey kidney cells to act as the target substrate for these recombination events. The SV40- and pBR322-derived plasmid DNA was irradiated with various doses of radiation before transfection into the transformed mammalian host cells. The successful homologous transfer of the bacterial ampicillin resistance (amp^{rm r}) gene from the inserted sequences to replace a mutant amp^->=ne on the shuttle vector was identified by plasmid extraction and transformation into E. coli host cells. Ultraviolet light (UV) was found not to induce homologous plasmid-chromosome recombination, while gamma radiation increased the frequency of recombinant plasmids detected. The introduction of specific double -strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was found to be increased by plasmid UV irradiation, but not to change with time. Plasmid survival, recombination, and mutagenesis were not affected by treating the mammalian host cells with UV light prior to plasmid transfection. The amp^{rm r} recombinant plasmid molecules analyzed were found to be mostly the result of nonconservative exchanges which appeared to involve both homologous and possibly nonhomologous interactions with the host chromosome. The observation that these recombinant structures were obtained from all of the plasmid alterations investigated suggests a common mechanistic origin for plasmid -chromosome recombination in these mammalian cells.

  7. Cytological Studies of Human Meiosis: Sex-Specific Differences in Recombination Originate at, or Prior to, Establishment of Double-Strand Breaks

    PubMed Central

    Gruhn, Jennifer R.; Rubio, Carmen; Broman, Karl W.; Hunt, Patricia A.; Hassold, Terry

    2013-01-01

    Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB) formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC) length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway. PMID:24376867

  8. Cohesin Is limiting for the suppression of DNA damage-induced recombination between homologous chromosomes.

    PubMed

    Covo, Shay; Westmoreland, James W; Gordenin, Dmitry A; Resnick, Michael A

    2010-07-01

    Double-strand break (DSB) repair through homologous recombination (HR) is an evolutionarily conserved process that is generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes. Here we show that the sister chromatid cohesion complex (cohesin) is a limiting factor in the control of DSB repair and genome stability and that it suppresses DNA damage-induced interactions between homologues. We developed a gene dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid cohesion subunit genes affected survival of gamma-irradiated G(2)/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G(2)/M cells with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage-induced recombinants in G(2)/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer.

  9. Meiotic sex chromosome inactivation.

    PubMed

    Turner, James M A

    2007-05-01

    X chromosome inactivation is most commonly studied in the context of female mammalian development, where it performs an essential role in dosage compensation. However, another form of X-inactivation takes place in the male, during spermatogenesis, as germ cells enter meiosis. This second form of X-inactivation, called meiotic sex chromosome inactivation (MSCI) has emerged as a novel paradigm for studying the epigenetic regulation of gene expression. New studies have revealed that MSCI is a special example of a more general mechanism called meiotic silencing of unsynapsed chromatin (MSUC), which silences chromosomes that fail to pair with their homologous partners and, in doing so, may protect against aneuploidy in subsequent generations. Furthermore, failure in MSCI is emerging as an important etiological factor in meiotic sterility.

  10. Meiotic chromosome abnormalities in human spermatogenesis.

    PubMed

    Martin, Renée H

    2006-08-01

    The last few years have witnessed an explosion in the information about chromosome abnormalities in human sperm and the meiotic events that predispose to these abnormalities. We have determined that all chromosomes are susceptible to nondisjunction, but chromosomes 21 and 22 and, especially, the sex chromosomes have an increased frequency of aneuploidy. Studies are just beginning on the effects of potential mutagens on the chromosomal constitution of human sperm. The effects of pesticides and cancer therapeutic agents have been reviewed. In the last decade, there has been a great impetus to study chromosome abnormalities in sperm from infertile men because the advent of intracytoplasmic sperm injection (ICSI) made it possible for these men to father pregnancies. A large number of studies have demonstrated that infertile men have an increased frequency of chromosomally abnormal sperm and children, even when they have a normal somatic karyotype. Meiotic studies on the pachytene stage of spermatogenesis have demonstrated that infertile men have impaired chromosome synapsis, a significantly decreased frequency of recombination, and an increased frequency of chromosomes completely lacking a recombination site. Such errors make these cells susceptible to meiotic arrest and the production of aneuploid gametes.

  11. Maintenance of Genome Stability and Breast Cancer: Molecular Analysis of DNA Damage-Activated Kinases

    DTIC Science & Technology

    2007-03-01

    286:1162-1166. 9. Ding, S. L., L. F. Sheu, J. C. Yu, T. L. Yang, B. F. Chen, F. J. Leu, and C. Y. Shen. 2004. Abnormality of the DNA double-strand...Soustelle, C., M. Vedel, R. Kolodner, and A. Nicolas. 2002. Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae

  12. Safety and Efficacy of a Recombinant DNA Plasmodium Falciparum Sporozoite Vaccine

    DTIC Science & Technology

    1987-06-06

    wells with serial dilutionsVolunteers were recruited under protocols approved by an of IgGI, lgG2, 1gG3, or IgG4 human myeloma proteins. IgE...measurement of antibodies directed s0 200 Dilution 60 0 3200 against the tets portion of R32tet, a related recombinant protein containing the identical CS... DISEASE IN PREGNANCY WITH A CLOSELY recombinant CS protein in FSV-1 consists of multiply LINKED DNA h &J2JE repeated units of only four aminoacids

  13. Replication factor A is required in vivo for DNA replication, repair, and recombination.

    PubMed Central

    Longhese, M P; Plevani, P; Lucchini, G

    1994-01-01

    Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta. Images PMID:7969128

  14. Dual recombinant Lactococcus lactis for enhanced delivery of DNA vaccine reporter plasmid pPERDBY.

    PubMed

    Yagnik, Bhrugu; Sharma, Drashya; Padh, Harish; Desai, Priti

    2017-03-04

    Food grade Lactococcus lactis (L. lactis) has been widely used as an antigen and DNA delivery vehicle. We had previously reported the use of non-invasive L. lactis for the delivery of newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, we outline the construction of dual recombinant L. lactis expressing Internalin A of Listeria monocytogenes and harbouring pPERDBY (LL InlA+ pPERDBY) to enhance the DNA delivery efficiency of L. lactis. After confirmation and validation of LL InlA+ pPERDBY, its DNA delivery potential was compared with previously developed non-invasive r- L. lactis::pPERDBY. The use of invasive L. lactis resulted in around three fold increase in number of Enhanced Green Fluorescent Protein expressing Caco- cells. Thus, these findings reinforce the prospective application of invasive strain of L. lactis in delivery of DNA/RNA and antigens.

  15. Mature cystic teratomas arise from meiotic oocytes, but not from pre-meiotic oogonia.

    PubMed

    Kaku, Hiroshi; Usui, Hirokazu; Qu, Jia; Shozu, Makio

    2016-04-01

    Mature cystic teratomas (MCTs) in the ovaries have been thought to originate from germ cells from all developmental stages, i.e., from pre-meiotic oogonia through meiotic oocytes to mature post-meiotic ova. This view was based on research on MCTs by classical methods, including those involving centromeric heteromorphisms in karyotypes, enzyme polymorphisms, and DNA polymorphisms. However, insufficient genomic information was obtained in those studies. The current study aimed to confirm the cytogenetic origin of ovarian MCTs by using short tandem repeat (STR) polymorphism analysis to obtain sufficient genomic information, especially in connection with centromeric loci. Tissue samples of MCTs (57 ovaries from 51 patients, 91 MCTs, 156 specimens in total) obtained from cystectomies or oophorectomies were used. We categorized the specimens into two groups: i) solid components of MCTs and ii) cyst walls. The numbers of solid components of MCTs from pre-meiotic oogonia, primary oocytes, secondary oocytes, and ova were 0, 33, 16, and 15, respectively. There were no pre-meiotic oogonia in this series of solid-component specimens. We propose a hypothesis for the tumorigenesis of ovarian MCTs: the precursors of ovarian MCTs are not functional oocytes or ova, but are primary oocytes that have escaped from meiotic arrest. This hypothesis could satisfactorily explain the lack of pre-meiotic teratomas observed in this study and the nearly equal distribution of teratomas originating from primary oocytes, secondary oocytes, and ova in previous studies. Furthermore, this hypothesis could provide a starting point for determining the mechanism underlying tumorigenesis of ovarian MCTs.

  16. DNA helicases in recombination and repair: construction of a delta uvrD delta helD delta recQ mutant deficient in recombination and repair.

    PubMed Central

    Mendonca, V M; Klepin, H D; Matson, S W

    1995-01-01

    DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair. They are involved in both the generation of recombinogenic single-stranded DNA ends and branch migration of synapsed Holliday junctions. Escherichia coli helicases II (uvrD), IV (helD), and RecQ (recQ) have all been implicated in the presynaptic stage of recombination in the RecF pathway. To probe for functional redundancy among these helicases, mutant strains containing single, double, and triple deletions in the helD, uvrD, and recQ genes were constructed and examined for conjugational recombination efficiency and DNA repair proficiency. We were unable to construct a strain harboring a delta recQ delta uvrD double deletion in a recBC sbcB(C) background (RecF pathway), suggesting that a delta recQ deletion mutation was lethal to the cell in a recBC sbcB(C) delta D background. However, we were able to construct a triple delta recQ delta uvrD Delta helD mutant in the recBC sbcB(C) background. This may be due to the increased mutator frequency in delta uvrD mutants which may have resulted in the fortuitous accumulation of a suppressor mutation(s). The triple helicase mutant recBC sbcB(C) delta uvrD delta recQ delta helD severely deficient in Hfr-mediated conjugational recombination and in the repair of methylmethane sulfonate-induced DNA damage. This suggests that the presence of at least one helicase--helicase II, RecQ helicase, or helicase IV--is essential for homologous recombination and recombinational DNA repair in a recBC sbcB(C) background. The triple helicase mutant was recombination and repair proficient in a rec+ background. Genetic analysis of the various double mutants unmasked additional functional redundancies with regard to conjugational recombination and DNA repair, suggesting that mechanisms of recombination depend both on the DNA substrates and on the genotype of the cell. PMID:7868608

  17. Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications

    PubMed Central

    Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

    2016-01-01

    Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination), umuCD (mutational DNA replication), as well as the key SOS genes lexA (regulation of the SOS system) and sulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA, and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively, these findings

  18. Control of Meiotic Crossovers: From Double-Strand Break Formation to Designation

    PubMed Central

    Gray, Stephen

    2017-01-01

    Meiosis, the mechanism of creating haploid gametes, is a complex cellular process observed across sexually reproducing organisms. Fundamental to meiosis is the process of homologous recombination, whereby DNA double-strand breaks are introduced into the genome and are subsequently repaired to generate either noncrossovers or crossovers. Although homologous recombination is essential for chromosome pairing during prophase I, the resulting crossovers are critical for maintaining homolog interactions and enabling accurate segregation at the first meiotic division. Thus, the placement, timing, and frequency of crossover formation must be exquisitely controlled. In this review, we discuss the proteins involved in crossover formation, the process of their formation and designation, and the rules governing crossovers, all within the context of the important landmarks of prophase I. We draw together crossover designation data across organisms, analyze their evolutionary divergence, and propose a universal model for crossover regulation. PMID:27648641

  19. Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

    PubMed Central

    Mosig, Gisela; Gewin, John; Luder, Andreas; Colowick, Nancy; Vo, Daniel

    2001-01-01

    Two major pathways of recombination-dependent DNA replication, “join-copy” and “join-cut-copy,” can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses. PMID:11459968

  20. A general method to modify BACs to generate large recombinant DNA fragments.

    PubMed

    Shen, Wei; Huang, Yue; Tang, Yi; Liu, De-Pei; Liang, Chih-Chuan

    2005-11-01

    Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the modification of BACs easy and reliable. We report here a modified recombineering method that can efficiently mediate the fusion of large DNA fragments from two or more different BACs. With the introduction of kanamycin-resistant gene and proposed rare-cutting restriction endonuclease (RCRE) sites into two BACs, a 82.6-kb DNA fragment containing the inverted human alpha-globin genes (theta, alpha1, alpha2, and zeta) from BAC191K2 and the locus control region (LCR) of human beta-globin gene locus (from the BAC186D7) was reconstructed. This approach for combining different BAC DNA fragments should facilitate many kinds of genomic experiments.

  1. Cytogenetic mapping with centromeric bacterial artificial chromosomes contigs shows that this recombination-poor region comprises more than half of barley chromosome 3H.

    PubMed

    Aliyeva-Schnorr, Lala; Beier, Sebastian; Karafiátová, Miroslava; Schmutzer, Thomas; Scholz, Uwe; Doležel, Jaroslav; Stein, Nils; Houben, Andreas

    2015-10-01

    Genetic maps are based on the frequency of recombination and often show different positions of molecular markers in comparison to physical maps, particularly in the centromere that is generally poor in meiotic recombinations. To decipher the position and order of DNA sequences genetically mapped to the centromere of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization with mitotic metaphase and meiotic pachytene chromosomes was performed with 70 genomic single-copy probes derived from 65 fingerprinted bacterial artificial chromosomes (BAC) contigs genetically assigned to this recombination cold spot. The total physical distribution of the centromeric 5.5 cM bin of 3H comprises 58% of the mitotic metaphase chromosome length. Mitotic and meiotic chromatin of this recombination-poor region is preferentially marked by a heterochromatin-typical histone mark (H3K9me2), while recombination enriched subterminal chromosome regions are enriched in euchromatin-typical histone marks (H3K4me2, H3K4me3, H3K27me3) suggesting that the meiotic recombination rate could be influenced by the chromatin landscape.

  2. H4K16 acetylation affects recombination and ncRNA transcription at rDNA in Saccharomyces cerevisiae.

    PubMed

    Cesarini, Elisa; D'Alfonso, Anna; Camilloni, Giorgio

    2012-07-01

    Transcription-associated recombination is an important process involved in several aspects of cell physiology. In the ribosomal DNA (rDNA) of Saccharomyces cerevisiae, RNA polymerase II transcription-dependent recombination has been demonstrated among the repeated units. In this study, we investigate the mechanisms controlling this process at the chromatin level. On the basis of a small biased screening, we found that mutants of histone deacetylases and chromatin architectural proteins alter both the amount of Pol II-dependent noncoding transcripts and recombination products at rDNA in a coordinated manner. Of interest, chromatin immunoprecipitation analyses in these mutants revealed a corresponding variation of the histone H4 acetylation along the rDNA repeat, particularly at Lys-16. Here we provide evidence that a single, rapid, and reversible posttranslational modification-the acetylation of the H4K16 residue-is involved in the coordination of transcription and recombination at rDNA.

  3. Robotics for recombinant DNA and human genetics research

    SciTech Connect

    Beugelsdijk, T.J.

    1990-01-01

    In October of 1989, molecular biologists throughout the world formally embarked on ultimately determining the set of genetic instructions for a human being. Called by some the Manhattan Project'' a molecular biology, pursuit of this goal is projected to require approximately 3000 man years of effort over a 15-year period. The Humane Genome Initiative is a worldwide research effort that has the goal of analyzing the structure of human deoxyribonucleic acid (DNA) and determining the location of all human genes. The Department of Energy (DOE) has designated three of its national laboratories as centers for the Human Genome Project. These are Los Alamos National Laboratory (LANL), Lawrence Livermore National Laboratory (LLNL), and Lawrence Berkeley Laboratory (LBL). These laboratories are currently working on different, but complementary technology development areas in support of the Human Genome Project. The robotics group at LANL is currently working at developing the technologies that address the problems associated with physical mapping. This article describes some of these problems and discusses some of the robotics approaches and engineering tolls applicable to their solution. 7 refs., 8 figs., 1 tab.